Methods for simultaneous control of lignin content and composition, and cellulose content in plants

ABSTRACT

The present invention relates to a method of concurrently introducing multiple genes into plants and trees is provided. The method includes simultaneous transformation of plants with multiple genes from the phenylpropanoid pathways including 4CL, CAld5H, AldOMT, SAD and CAD genes and combinations thereof to produce various lines of transgenic plants displaying altered agronomic traits. The agronomic traits of the plants are regulated by the orientation of the specific genes and the selected gene combinations, which are incorporated into the plant genome.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/230,086, filed on Sep. 5, 2000, and is incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with United States government supportawarded by the Energy Biosciences Program, United States Department ofEnergy, and the United States Department of Agriculture research grantnumbers USDA 99-35103-7986, USDA 01-03749, and DOE DE-FG02-01ER15179.The United States government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] The invention provides a method of introducing two or more genes,involved in lignin biosynthesis, into plant cells. The method of theinvention employs either an Agrobacterium-mediated or other appropriateplant gene delivery system by which multiple genes together with asingle selectable marker gene are simultaneously transferred andinserted into the genome of plants with high frequencies.

[0004] The ability to introduce foreign genes into plants is aprerequisite for engineering agronomic traits in plants. Many systemshave been developed for introducing a foreign gene into plant cells,which involve mainly either Agrobacterium- or microprojectilebombardment-mediated transformation (Christou, 1996). The principle ofall these systems involves the insertion of a target gene into the hostplant genome together with a selectable marker gene encoding eitherantibiotic or herbicide resistance to aid in the selection of transgeniccells from non-transgenic cells. These systems generally are onlyeffective for introducing a single target gene into the host plant.

[0005] To alter agronomic traits, which generally are polygenic innature, multiple genes involved in complex biosynthetic pathways must beintroduced and expressed in plant cells. In this context, thetraditional single-gene transfer systems are essentially useless for thefollowing two reasons: 1) it is impractical to introduce multiple genesby repetitive insertion of single genes into transgenic plants due tothe time and effort required for recovery of the transgenic tissues; inparticular, a repetitive single-gene approach is highly impractical forplant species such as trees which, depending upon the species, requiretwo to three years for transgenic tissue selection and regeneration intoa tree; and 2) the presence of a selectable marker gene in a transgenicline precludes the use of the same marker gene in subsequenttransformations of plant material from that line. Moreover, the numberof available marker genes is limited, and many plant species arerecalcitrant to regeneration unless appropriate antibiotic or herbicideselection is used.

[0006] Chen et aL (1998) recently reported the genetic transformation ofrice with multiple genes by cobombardment of several gene constructsinto embryogenic suspension tissues. However, particlebombardment-mediated gene transfer into embryogenic tissues is highlyspecies-dependent, and regeneration of whole plants from embryogeniccells cannot be achieved for a variety of plant species (Horsch et al.,1985).

[0007] In contrast, Agrobacterium-mediated gene transfer and whole plantregeneration through organogenesis is a simple process and a lessspecies-dependent system than bombardment-mediated transformation andregeneration via embryogenesis. However, the introduction of more thanone gene in a single plasmid vector via Agrobacterium may be technicallytroublesome and limited by the number or the size of the target genes(Chen et al., 1998). For example, Tricoli et al. (1995) reported thetransfer of three target genes to squash via Agrobacterium-mediated genetransfer. A binary plasmid vector containing the three target genes wasincorporated into an Agrobacterium strain, which was subsequently usedto infect the leaf tissue of squash. As only one line was recovered fromnumerous infected squash tissues that contained all of the target genes,the use of a single binary vector with a number of genes appears to be ahighly inefficient method to produce transgenic plants with multiplegene transfers. Therefore, it was commonly accepted that transfer ofmultiple genes via Agrobacterium-mediated transformation was impractical(Ebinuma et al., 1997), until success of multiple gene transfer viaAgrobacterium was first reported in co-pending, commonly owned PCTapplication, PCT/US/0027704, filed Oct. 6, 2000, entitled “Method ofIntroducing a Plurality of Genes into Plants” by Chiang et al,incorporated herein by reference. However, homologous tissue-specificpreparation of transgenic trees to specifically alter lignin content,increase S/G (syringyl:guaiacyl) lignin ratio and increase cellulosequantity, as compared to an untransformed plant was unsuccessful.

[0008] Yet, the altering of lignin content and composition in plants hasbeen a goal of genetically engineered traits in plants. Lignin, acomplex phenolic polymer, is a major part of the supportive structure ofmost woody plants including angiosperm and gymnosperm trees, which, inturn, are the principal sources of fiber for making paper and cellulosicproducts. Lignin generally constitutes about 25% of the dry weight ofthe wood, making it the second most abundant organic compound on earthafter cellulose. Lignin provides rigidity to wood for which it is wellsuited due, in part, to its resistance to biochemical degradation.

[0009] Despite its importance to plant growth and structure, lignin isnonetheless problematic to post-harvest, cellulose-based wood/cropprocessing for fiber, chemical, and energy production because it must beremoved or degraded from cellulose at great expense. Certain structuralconstituents of lignin, such as the guaiacyl (G) moiety, promote monomercross-linkages that increase lignin resistance to degradation (Sarkanen,1971; Chang and Sarkanen, 1973; Chiang and Funaoka, 1990). Inangiosperms, lignin is composed of a mixture of guaiacyl (G) andsyringyl (S) monolignols, and can be degraded at considerably lessenergy and chemical cost than gymnosperm lignin, which consists almostentirely of guaiacyl moieties (Freudenberg, 1965). It has been estimatedthat, if syringyl lignin could be genetically incorporated intogymnosperm guaiacyl lignin or into angiosperms to increase the syringyllignin content, the annual saving in processing of such geneticallyengineered plants as opposed to their wild types would be in the rangeof $6 to $10 billion in the U.S. alone. Consequently, there has beenlong-standing incentive to understand the biosynthesis of syringylmonolignol to genetically engineer plants to contain more syringyllignin, thus, facilitating wood/crop processing (Trotter, 1990; Bugos etal., 1991; Boudet et al., 1995; Hu et al., 1999).

[0010] Depending on the use for the plant, genetic engineering ofcertain traits has been attempted. For some plants, as indicated above,there has been a long-standing incentive to genetically modify ligninand cellulose to decrease lignin and increase cellulose contents. Forexample, it has been demonstrated that the digestibility of forage cropsby ruminants is inversely proportional to lignin content in plants(Buxton and Roussel, 1988, Crop. Sci., 28, 553-558; Jung and Vogel,1986, J. Anim., Sci., 62, 1703-1712). Therefore, decreased lignin andhigh cellulose plants are desirable in forage crops to increase theirdigestibility by ruminants, thereby providing the animal with morenutrients per unit of forage.

[0011] In other plants, genetically increasing the S/G ratio of thelignin has been sought. As noted above, lignin in angiosperms iscomposed of guaiacyl (G) and syringyl (S) monomeric units, whereasgymnosperm lignin consists entirely of G units. The structuralcharacteristics of G units in gymnosperm lignin promote monomercross-linkages that increase lignin resistance to chemical extractionduring wood pulp production. However, the S units present in angiospermlignin prevent such chemical resistant cross-links. Therefore, withoutexception, chemical extraction of G lignin in pulping of gymnosperms ismore difficult and requires more chemicals, longer reaction times andhigher energy levels than the extraction of G-S lignin during pulping ofangiosperms (Sarkanen, K. V., 1971, in Lignins: Occurrence, Formation,Structure and Reaction, Sarkanen, K. V. & Ludwig, C. H., eds.,Wiley-Interscience, New York; Chang, H. M. and Sarkanen, K. V., 1973,TAPPI, 56:132-136). As a rule, the reaction rate of extracting ligninduring wood pulping is directly proportional to the quantity of the Sunit in lignin (Chang, H. M. and Sarkanen, K. V., 1973, TAPPI,56:132-136). Hence, altering lignin into more reactive G-S type ingymnosperms and into high S/G ratio in angiosperms would represent apivotal opportunity to enhance current pulping and bleaching efficiencyand to provide better, more economical, and more environmentally soundutilization of wood.

[0012] Recent results have indicated that high S/G ratio may also addfurther mechanical advantages to plants, balancing the likely loss ofsturdiness of plants with severe lignin reduction (Li et al., 2001,Plant Cell, 13:1567-1585). Moreover, a high S/G lignin ratio would alsoimprove the digestibility of forage crops by ruminants (Buxton andRoussel, 1988, Crop. Sci., 28, 553-558; Jung and Vogel, 1986, J. Anim.,Sci., 62, 1703-1712).

[0013] In some applications, both a high lignin content and high S/Gratio have been sought (i.e., combining these two traits in plants). Forexample, it has been demonstrated that when lignin is extracted out fromwood during chemical pulping, lignin in the pulping liquor is normallyused as a fuel source to provide energy to the pulping and bleachingoperations. This lignin-associated energy source, which is not necessaryfor pulp mills using purchased fuel for energy, is essential to somepulp mills which depend upon internal sources, such as extracted lignin,to be self-sufficient in energy. Therefore, for this purpose, it may bedesirable to increase lignin content in pulpwood species, and at thesame time to increase the S/G ratio in these species to facilitate theextraction of more lignin to be used as fuel.

[0014] Additionally, for grain production and other non-relatedpurposes, increased lignin content and/or S/G lignin ratio are desirableto provide extra sturdiness in plants to prevent the loss of sociallyand economically important food crops due to dislodging and due todamage to the aerial parts of the plant.

[0015] The plant monolignol biosynthetic pathway is set forth in FIG. 1and will be explained in more detail hereinbelow. The key lignin controlsites in the monolignol biosynthetic pathway are mediated by genesencoding the enzymes 4-coumarate-CoA ligase (4CL) (Lee et al., 1997),coniferyl aldehyde 5-hydroxylase (CAld5H) (Osakabe et al., 1999) andS-adenosyl-L-methionine (SAM)-dependent 5-hydroxyconiferaldehydeO-methyltransferase (AldOMT) (Li et al., 2000), respectively, for theformation of sinapaldehyde (see, FIG. 1). Further, coniferyl alcoholdehydrogenase (CAD) (MacKay et al., 1997) catalyzes the reactionincluding the substrate coniferaldehyde to coniferyl alcohol. It hasrecently been discovered that sinapyl alcohol dehydrogenase (SAD)enzymatically converts sinapaldehyde into sinapyl alcohol, the syringylmonolignol, for the biosynthesis of syringyl lignin in plants (see, FIG.1). See, concurrently filed, commonly owned U.S. non-provisionalapplication entitled “Genetic Engineering of Syringyl-Enriched Lignin inPlants,” incorporated herein by reference. It should be noted that thegene encoding the enzyme sinapyl alcohol dehydrogenase (SAD) representsthe last gene that is indispensable for genetic engineering of syringyllignin in plants.

[0016] A summary of the conserved regions contained within the codingsequence of each of the above listed proteins is described below.Because SAD is a recently discovered enzyme in Aspen, sequencealignments with other representative species were unable to beperformed.

[0017] The protein sequence alignments of plant AldOMTs are shown inFIG. 9. All AldOMTs have three conserved sequence motifs (I, II, andIII) which are the binding sites of S-adenosyl-L-methionine (SAM), theco-substrate or methyl donor for the OMT reaction (Ibrahim, 1997, TrendsPlant Sci., 2:249-250; Li et al., 1997, Proc. Natl. Acad. Sci. USA,94:5461-5466; Joshi and Chiang, 1998, Plant Mol. Biol., 37:663-674).These signature sequence motifs and the high sequence homology of theseproteins to PtAldOMT attest to their function as an AldOMT specific forconverting 5-hydroxyconiferaldehyde into sinapaldehyde (Li et al., 2000,J. Biol. Chem., 275:6537-6545). This AldOMT, like CAld5H, also operatesat the aldehyde level of the plant monolignol biosynthetic pathway.

[0018] The protein sequence alignments of plant CADs are shown in FIG.10. It was recently proven that CADs are actually guaiacyl monolignolpathway specific (Li et al., 2001, Plant Cell, 13:1567-1585). Based onhigh sequence homology, the alignment program picked up CADs fromangiosperms as well as gymnosperms (radiata pine, loblolly pine andspruce) which have only G-lignin. All CADs have the Zn1 binding motifand structural Zn2 consensus region, as well as a NADP binding site(Jornvall et al., 1987, Eur. J. Biochem., 167:195-201; MacKay et al.,1995, Mol. Gen. Genet., 247:537-545). All these sequence characteristicsand high sequence homology to PtCAD attest to these CAD function as aG-monolignol specific CAD (Li et al., 2001, Plant Cell, 13:1567-1585).

[0019] The protein sequence alignments of plant Cald5Hs are shown inFIG. 11. Although, there are different types of 5-hydroxylases, i.e.,F5H, CAld5H is the sole enzyme catalyzing specifically the conversion ofconiferaldehyde into 5-hydroxyconiferaldehyde. All full-length CAld5Hshave the proline-rich region located from amino acid 40 to 45 which isbelieved to be involved in the process of correct folding of microsomalP450s and is also important in heme incorporation into P450s (Yamazakiet al. 1993, J. Biochem. 114:652-657). Also they all have theheme-binding domain (PFGXGXXXCXG) that is conserved in all P450 proteinsNelson et al. 1996, Pharmacogenetics, 6:1-41). These signature sequencesand the high sequence homology of these proteins to PtCAld5H theirfunction as a 5-hydroxylase that is specific for convertingconiferaldehyde into 5-hydroxyconiferaldehyde (Osakabe et al., 1999,Proc. Natl. Acad. Sci. USA, 96:8955-8960).

[0020] The protein sequence alignment of plant 4CLs are shown in FIG.12. In general, 4CL catalyzes the activation of the hydroxycinnamicacids to their corresponding hydroxycinnamoyl-CoA esters. 4CL has thehighest activity with ρ-coumaric acid. 4CL cDNA sequences have beenreported from a number of representative angiosperms and gymnosperms,revealing two highly conserved regions, a putative AMP-binding region(SSGTTGLPKGV), and a catalytic motif (GEICIRG). The amino acid sequencesof 4CL from plants contain a total of five conserved Cys residues.

[0021] Despite recognition of these key enzymes in lignin biosynthesis,there continues to be a need to develop an improved method tosimultaneously control the lignin quantity, lignin compositions, andcellulose contents in plants by introducing multiple genes into plantcells.

BRIEF SUMMARY OF THE INVENTION

[0022] The invention provides a method of introducing two or more genesinvolved in lignin biosynthesis present in one or more independentvectors into plant cells. The method of the invention suitably employsan Agrobacterium-mediated or another gene delivery system by whichmultiple genes together with a single selectable marker gene aresimultaneously transferred and inserted into the genome of plants withhigh frequencies.

[0023] If an Agrobacterium-mediated gene delivery system is used, eachgene of interest is present in a binary vector that has been introducedinto Agrobacterium to yield an isolated Agrobacterium strain comprisingthe binary vector. Moreover, more than one gene of interest may bepresent in each binary vector. Plant materials comprising plant cells,e.g., plant seed, plant parts or plant tissue including explantmaterials such as leaf discs, from a target plant species are suitablyinoculated with at least two, preferably at least three, and morepreferably at least four or more, of the isolated Agrobacterium strains,each containing a different gene of interest. A mixture of the strainsis suitably contacted with plant cells. At least one of the binaryvectors in the isolated Agrobacterium strains contains a marker gene,and any marker gene encoding a trait for selecting transformed cellsfrom non-transformed cells may be used. Transformed plant cells areregenerated to yield a transgenic plant, the genome of which isaugmented with DNA from at least two, preferably at least three, andmore preferably at least four, and even more preferably at least five ofthe binary vectors.

[0024] The method of the invention is thus applicable to all plantspecies that are susceptible to the transfer of genetic information byAgrobacterium or other gene delivery system. Suitable plant speciesuseful in the method of the invention include agriculture and foragecrops, as well as monocots. In particular, plant species useful in themethod of the invention include trees, e.g., angiosperms andgymnosperms, and more suitably a forest tree, but are not limited to thetree.

[0025] The method of the invention is suitably employed to enhance adesired agronomic trait by altering the expression of two or more genes.Such traits include alterations in lignin biosynthesis (e.g., reduction,augmentation and/or structural changes), cellulose biosynthesis (e.g.,augmentation, reduction, and/or quality including high degree ofpolymerization and crystallinity), growth, wood quality (e.g., highdensity, low juvenile wood, high mature wood, low reaction wood,desirable fiber angle), stress resistance (e.g., cold-, heat-, andsalt-tolerance, pathogen-, insect- and other disease-resistance,herbicide-resistance), sterility, high grain yield (for forage and foodcrops), and increased nutrient level.

[0026] Thus, the present invention advantageously provides gymnospermand angiosperm plants with decreased lignin content, increasedsyringyl/guaiacyl (S/G) lignin ratio and increased cellulose content inwhich a single trait or multiple traits are changed.

[0027] In another aspect, the invention provides gymnosperm plants withsyringyl enriched lignin and/or increased lignin content and/orincreased syringyl/guaiacyl (S/G) lignin ratio.

[0028] Similarly, the present invention also provides angiosperm plantswith increased lignin content.

[0029] Other advantages and a fuller appreciation of specific attributesand variations of the invention will be gained upon an examination ofthe following detailed description of exemplary embodiments and the likein conjunction with the appended claims.

BRIEF DESCRIPTION OF THE DRAWING(S)

[0030]FIG. 1 is a schematic representation of plant monolignol pathwaysfor production of coniferyl alcohol and sinapyl alcohol;

[0031]FIG. 2 is the SAD polynucleotide DNA sequence (SEQ ID NO: 1) andthe SAD amino acid sequence (SEQ ID NO: 2) respectively FIG. 2A and 2B;

[0032]FIG. 3 is the CAld5H polynucleotide DNA sequence (SEQ ID NO: 3)and the CAld5H amino acid sequence (SEQ ID NO: 4) respectively FIG. 3Aand 3B;

[0033]FIG. 4 is the AldOMT polynucleotide DNA sequence (SEQ ID NO: 5)and the AldOMT amino acid sequence (SEQ ID NO: 6) respectively FIG. 4Aand 4B;

[0034]FIG. 5 is the 4CL polynucleotide DNA sequence (SEQ ID NO: 7) andthe 4CL amino acid sequence (SEQ ID NO: 10) respectively FIG. 5A and 5B;

[0035]FIG. 6 is the CAD polynucleotide DNA sequence (SEQ ID NO: 8) andthe CAD amino acid sequence (SEQ ID NO: 9) respectively FIG. 6A and 6B;

[0036]FIG. 7 is a map of the DNA construct, pBKPpt_(4CL) Pt4CL1-a,positioned in a plant transformation binary vector.

[0037]FIG. 8 is a map of the DNA construct, pBKPpt_(4CL) PtCAld5H-s,positioned in a plant transformation binary vector.

[0038]FIG. 9 is the protein sequence alignment of AldOMTs forrepresentative species of plants.

[0039]FIG. 10 is the protein sequence alignment of CADs forrepresentative species of plants.

[0040]FIG. 11 is the protein sequence alignment of CAld5Hs forrepresentative species of plants.

[0041]FIG. 12 is the protein sequence alignment of 4CLs forrepresentative species of plants.

[0042] It is expressly understood that the figures of the drawing arefor the purposes of illustration and description only and are notintended as a definition of the limits of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention provides a method and DNA constructs usefulfor the transformation of plant tissue for the alteration of ligninmonomer composition, increased syringyl/guaiacyl (S/G) lignin ratio andincreased cellulose content and transgenic plants resulting from suchtransformations. The present invention is of particular value to thepaper and pulp industries because lignin containing higher syringylmonomer content is more susceptible to chemical delignification. Woodyplants transformed with the DNA constructs provided herein offer asignificant advantage in the delignification process over conventionalpaper feedstocks. Similarly, modification of the lignin composition ingrasses by the insertion and expression of a heterologous SAD geneoffers a unique method for increasing the digestibility of grasses andis of significant potential economic benefit to the farm andagricultural industries.

[0044] The terms used in this specification generally have theirordinary meanings in the art, within the context of the invention and inthe specific context where each term is used. Certain terms arediscussed below, or elsewhere in the specification, to provideadditional guidance to the person of skill in the art in describing thecompositions and methods of the invention and how to make and use them.It will be appreciated that the same thing can be said in more than oneway. Consequently, alternative language and synonyms may be used for anyone or more of the terms discussed herein, nor is any specialsignificance to be placed upon whether or not a term is elaborated ordiscussed herein. Synonyms for certain terms are provided. A recital ofone or more synonyms does not exclude the use of other synonyms. The useof examples anywhere in this specification, including examples of anyterms discussed herein, is illustrative only, and in no way limits thescope and meaning of the invention or of any exemplified term. Likewise,the invention is not limited to the preferred embodiments.

[0045] As used herein, “gene” refers to a nucleic acid fragment thatexpresses a specific protein including the regulatory sequencespreceding (5′ noncoding) and following (3′ noncoding) the coding regionor coding sequence (See, below). “Native” gene refers to the gene asfound in nature with its own regulatory sequences.

[0046] “Endogenous gene” refers to the native gene normally found in itsnatural location in the genome.

[0047] “Transgene” refers to a gene that is introduced by gene transferinto the host organism.

[0048] “Coding sequence” or “Coding Region” refers to that portion ofthe gene that contains the information for encoding a polypeptide. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, for example, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA, and even syntheticDNA sequences.

[0049] “Promoter” or “Promoter Sequence” refers to a DNA sequence, in agiven gene, which sequence controls the expression of the codingsequence by providing the recognition site for RNA polymerase and otherfactors required for proper transcription. Most genes have regions ofDNA sequence that are promoter sequences which regulate gene expression.Promoter regions are typically found in the 5′ flanking DNA sequenceupstream from the coding sequence in both prokaryotic and eukaryoticcells. A promoter sequence provides for regulation of transcription ofthe downstream gene sequence and typically includes from about 50 toabout 2000 nucleotide base pairs. Promoter sequences also containregulatory sequences such as enhancer sequences that can influence thelevel of gene expression. Some isolated promoter sequences can providefor gene expression of heterologous DNAs, that is DNA different from thenatural homologous DNA. Promoter sequences are also known to be strongor weak or inducible. A strong promoter provides for a high level ofgene expression, whereas a weak promoter provides for a very low levelof gene expression. An inducible promoter is a promoter that providesfor turning on and off of gene expression in response to an exogenouslyadded agent or to an environmental or developmental stimulus. Anisolated promoter sequence that is a strong promoter for heterologousDNAs is advantageous because it provides for a sufficient level of geneexpression to allow for easy detection and selection of transformedcells, and provides for a high level of gene expression when desired. Apromoter may also contain DNA sequences that are involved in the bindingof protein factors which control the effectiveness of transcriptioninitiation in response to physiological or developmental conditions.

[0050] “Regulatory sequence(s)” refers to nucleotide sequences locatedupstream (5′), within, and/or downstream (3′) of a coding sequence,which control the transcription and/or expression of the codingsequences in conjunction with the protein biosynthetic apparatus of thecell. Regulatory sequences include promoters, translation leadersequences, transcription termination sequences and polyadenylationsequences.

[0051] “Encoding” and “coding” refer to the process by which a gene,through the mechanisms of transcription and translation, provides theinformation to a cell from which a series of amino acids can beassembled into a specific amino acid sequences to produce an activeenzyme. It is understood that the process of encoding a specific aminoacid sequence includes DNA sequences that may involve base changes thatdo not cause a change in the encoded amino acid, or which involve basechanges which may alter one or more amino acids, but do not affect thefunctional properties of the protein encoded by the DNA sequence. It istherefore understood that the invention encompasses more than thespecific exemplary sequences. Modifications to the sequences, such asdeletions, insertions or substitutions in the sequence which producesilent changes that do not substantially affect the functionalproperties of the resulting protein molecule are also contemplated. Forexample, alterations in the gene sequence which reflect the degeneracyof the genetic code, or which result in the production of a chemicallyequivalent amino acid at a given site, are contemplated. Thus, a codonfor the amino acid alanine, a hydrophobic amino acid, may be substitutedby a codon encoding another less hydrophobic residue, such as glycine,or a more hydrophobic residue, such as valine, leucine or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a biologically equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the protein molecule would also not be expectedto alter the activity of the protein. In some cases, it may in fact bedesirable to make mutants of the sequence to study the effect ofretention of biological activity of the protein. Each of these proposedmodifications is well within the routine skill in the art, as is thedetermination of retention of biological activity in the encodedproducts. Moreover, the skilled artisan recognizes that sequencesencompassed by this invention are also defined by their ability tohybridize, under stringent condition, with the sequences exemplifiedherein.

[0052] “Expression” is meant to refer to the production of a proteinproduct encoded by a gene. “Overexpression” refers to the production ofa gene product in transgenic organisms that exceed levels of productionin normal or non-transformed organisms.

[0053] “Functional portion” or “functional fragment” or “functionalequivalents” of an enzyme is that portion, fragment or equivalentsection which contains the active site for binding one or more reactantsor is capable of improving or regulating the rate of reaction. Theactive site may be made up of separate portions present on one or morepolypeptide chains and will generally exhibit high substratespecificity.

[0054] “Enzyme encoded by a nucleotide sequence” includes enzymesencoded by a nucleotide sequence which includes partial isolated DNAsequences.

[0055] “Transformation” refers to the transfer of a foreign gene intothe genome of a host organism and its genetically stable inheritance.

[0056] “% identity” refers to the percentage of the nucleotides/aminoacids of one polynucleotide/polypeptide that are identical to thenucleotides/amino acids of another sequence ofpolynucleotide/polypeptide as identified by a program such as GAP fromGenetics Computer Group Wisconsin (GCG) package (version 9.0) (Madison,Wis.). GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol.48:443-453, 1970) to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. Whenparameters required to run the above algorithm are not specified, thedefault values offered by the program are contemplated.

[0057] “Substantial homology” or “substantial similarity” refers to a70% or more similarity or 70% homology wherein “% similarity” or “%homology” between two polypeptide sequences is a function of the numberof similar positions shared by two sequences on the basis of the scoringmatrix used divided by the number of positions compared and thenmultiplied by 100. This comparison is made when two sequences arealigned (by introducing gaps if needed) to determine maximum homology.The PowerBlast program, implemented by the National Center forBiotechnology Information, can be used to compute optimal, gappedalignments. GAP program from Genetics Computer Group Wisconsin package(version 9.0) (Madison, Wis.) can also be used.

[0058] “Lignin monomer composition” refers to the relative ratios ofguaiacyl monomer and syringyl monomer found in lignified plant tissue.

[0059] “Plant” includes whole plants and portions of plants, includingplant organs (e.g., roots, stems, leaves, etc).

[0060] “Angiosperm” refers to plants that produce seeds encased in anovary. A specific example of an angiosperm is Liquidambar styraciflua(L.)[sweetgum].

[0061] “Gymnosperm” refers to plants that produce naked seeds, i.e.,seeds that are not encased in an ovary. A specific example of agymnosperm is Pinus taeda (L.)[loblolly pine].

[0062] As used herein, the terms “isolated and/or purified” withreference to a nucleic acid molecule or polypeptide refer to in vitroisolation of a nucleic acid or polypeptide molecule from its naturalcellular environment, and from association with other components of thecell, such as nucleic acid or polypeptide, so that it can be sequenced,replicated and/or expressed.

[0063] An “isolated” strain of Agrobacterium refers to cells derivedfrom a clone of Agrobacterium that is transformed in vitro with anisolated binary vector.

[0064] A “vector” is a recombinant nucleic acid construct, such asplasmid, phage genome, virus genome, cosmid, or artificial chromosome towhich a polynucleotide in accordance with the invention may be attached.In a specific embodiment, the vector may bring about the replication ofthe attached segment, e.g., in the case of a cloning vector.

[0065] “Sinapyl alcohol dehydrogenase” or “SAD”, coniferyl alcoholdehydrogenase or “CAD”, coniferaldeyde 5-hydroxylase or “Cald5H”,5-hydroxyconiferaldehyde O-methyltransferase or “AldOMT”, and4-coumarate-CoA ligase or “4CL” refer to enzymes in the plantphenylpropanoid biosynthetic pathway. In the illustrated embodiments ofthe present invention, the DNA sequences encoding these enzymes wereidentified from quaking aspen Populus tremuloides. It is understood thateach sequence can be used as a probe to clone its equivalent from anyplant species by techniques (EST, PCR, RT-PCR, antibodies, etc.) wellknown in the art.

[0066] The Phenyl Propanoid Biosynthetic Pathway

[0067] Reference is made to FIG. 1 which shows different steps in thebiosynthetic pathways from 4-coumarate (1) to guaiacyl (coniferylalcohol (6)) and syringyl (sinapyl alcohol (9)) monolignols for theformation of guaiacyl-syringyl lignin together with the enzymesresponsible for catalyzing each step. The enzymes indicated for each ofthe reaction steps are: 4-coumaric acid 3-hydroxylase (C3H) whichconverts 4-coumarate (1) to caffeate (2); 4-coumarate-CoA ligase (4CL)converts caffeate (2) to caffeoyl CoA (3) which in turn is converted toferuloyl CoA (4) by caffeoyl-CoA O-methyltransferase (CCoAOMT);cinnamoyl-CoA reductase (CCR) converts feruloyl CoA (4) toconiferaldehyde (5); coniferyl alcohol dehydrogenase (CAD) convertsconiferaldehyde (5) to the guaiacyl monolignol coniferyl alcohol (6); atconiferaldehyde (5), the pathway splits wherein coniferaldehyde (5) canalso be converted to 5-hydroxyconiferaldehyde (7) by coniferaldeyde5-hydroxylase (Cald5H); 5-hydroxyconiferaldehyde O-methyltransferase(AldOMT) converts 5-hydroxconiferaldehyde (7) to sinapaldehyde (8)which, in turn, is converted to the syringyl monolignol, sinapyl alcohol(9) by sinapyl alcohol dehydrogenase (SAD).

[0068] DNA Constructs

[0069] According to the present invention, a DNA construct is providedwhich is a plant DNA having a promoter sequence, a coding region and aterminator sequence. The coding region encodes a combination of enzymesessential to lignin biosynthesis, specifically, SAD, CAD, Cald5H,AldOMT, and 4CL protein sequences, substantially similar sequences, orfunctional fragments thereof. The coding region is suitably a minimumsize of 50 bases. The gene promoter is positioned at the 5′-end of atransgene (e.g., 4CL alone or together with SAD, Cald5H, and AldOMT, andcombinations thereof, or 4CL and CAD alone, or together with CAld5H,SAD, and AldOMT, and combinations thereof, as described hereinafter) forcontrolling the transgene expression, and a gene termination sequencethat is located at the 3′-end of the transgene for signaling the end ofthe transcription of the transgene.

[0070] The DNA construct in accordance with the present invention can beincorporated into the genome of a plant by transformation to alterlignin biosynthesis, increase syringyl/guaiacyl (S/G) lignin ratio andincrease cellulose content. The DNA construct may include clones ofCAld5H, SAD, AldOMT, CAD, and 4CL, and variants thereof such as arepermitted by the degeneracy of the genetic code and the functionalequivalents thereof.

[0071] The DNA constructs of the present invention may be inserted intoplants to regulate production the following enzymes: CAld5H, SAD,AldOMT, CAD, and 4CL. Depending on the nature of the construct, theproduction of the protein may be increased or decreased, eitherthroughout or at particular stages in the life of the plant, relative toa similar control plant that does not incorporate the construct into itsgenome. For example, the orientation of the DNA coding sequence,promoter, and termination sequence can serve to either suppress ligninformation or amplify lignin formation. For the down-regulation of ligninsynthesis, the DNA is in the antisense orientation. For theamplification of lignin biosynthesis, the DNA is in the senseorientation, thus to provide one or more additional copies of the DNA inthe plant genome. In this case, the DNA is suitably a full-length cDNAcopy. It is also possible to target expression of the gene to specificcell types of the plants, such as the epidermis, the xylem, the roots,etc. Constructs in accordance with the present invention may be used totransform cells of both monocotyledons and dicotyledons plants invarious ways known in the art. In many cases, such plant cells may becultured to regenerate whole plants which subsequently reproduce to givesuccessive generations of genetically modified plants. Examples ofplants that are suitably genetically modified in accordance with thepresent invention, include but are not limited to, trees such a aspen,poplar, pine and eucalyptus.

[0072] Promoters and Termination Sequences

[0073] Various gene promoter sequences are well known in the art and canbe used in the DNA constructs of present invention. The promoter in theconstructs in accordance with the present invention suitably providesfor expression of the linked DNA segment. The promoter can also beinducible so that gene expression can be turned on or off by anexogenously added agent. It may also be preferable to combine thedesired DNA segment with a promoter that provides tissue specificexpression or developmentally regulated gene expression in plants.

[0074] The promoter may be selected from promoters known to operate inplants, e.g., CaMV35S, GPAL2, GPAL3 and endogenous plant promotercontrolling expression of the enzyme of interest. Use of a constitutivepromoter such as the CaMV35S promoter (Odell et al. 1985), or CaMV 19S(Lawton et al., 1987) can be used to drive the expression of thetransgenes in all tissue types in a target plant. Other promoters arenos (Ebert et al. 1987), Adh (Walker et al., 1987), sucrose synthase(Yang et al., 1990), Δ-tubulin, ubiquitin, actin (Wang et al., 1992),cab (Sullivan et al., 1989), PEPCase (Hudspeth et al., 1989) or thoseassociate with the R gene complex (Chandler et al., 1989). On the otherhand, use of a tissue specific promoter permits functions to becontrolled more selectively. The use of a tissue-specific promoter hasthe advantage that the desired protein is only produced in the tissue inwhich its action is required. Suitably, tissue-specific promoters, suchas those would confine the expression of the transgenes in developingxylem where lignification occurs, may be used in the inventive DNAconstructs.

[0075] A DNA segment can be combined with the promoter by standardmethods as described in Sambrook et al., 2nd ed. (1982). Briefly, aplasmid containing a promoter such as the CaMV 35S promoter can beconstructed as described in Jefferson (1987) or obtained from ClontechLab, Palo Alto, Calif. (e.g., pBI121 or pBI221). Typically, theseplasmids are constructed to provide for multiple cloning sites havingspecificity for different restriction enzymes downstream from thepromoter. The DNA segment can be subcloned downstream from the promoterusing restriction enzymes to ensure that the DNA is inserted in properorientation with respect to the promoter so that the DNA can beexpressed.

[0076] The gene termination sequence is located 3′ to the DNA sequenceto be transcribed. Various gene termination sequences known in the artmay be used in the present inventive constructs. These include nopalinesynthase (NOS) gene termination sequence (see, e.g., references cited inco-pending, commonly-owned PCT application, PCT/US/0027704, filed Oct.6, 2000, entitled “Method of Introducing a Plurality of Genes intoPlants,” incorporated herein by reference.)

[0077] Marker Genes

[0078] A marker gene may also be incorporated into the inventive DNAconstructs to aid the selection of plant tissues with positiveintegration of the transgene. “Marker genes” are genes that impart adistinct phenotype to cells expressing the marker gene, and thus, allowsuch transformed cells to be distinguished from cells that do not havethe marker. Many examples of suitable marker genes are known to the artand can be employed in the practice of the invention, such as neomycinphosphotransferase II (NPT II) gene that confers resistance to kanamycinor hygromycin antibiotics which would kill the non-transformed planttissues containing no NPT II gene (Bevan et al., 1983). Numerous otherexemplary marker genes used in the method, in accordance with thepresent invention are listed in Table 1 of co-pending, commonly owned ofPCT/US/0027704, filed Oct. 6, 2000, entitled “Method of Introducing aPlurality of Genes into Plants,” incorporated herein by reference.

[0079] Therefore, it will be understood that the following discussion isexemplary rather than exhaustive. In light of the techniques disclosedherein and the general recombinant techniques which are known in theart, the present invention renders possible the introduction of anygene, including marker genes, into a recipient cell to generate atransformed plant.

[0080] Optional Sequences in the Expression Cassette

[0081] The expression cassette containing DNA sequences in accordancewith the present invention can also optionally contain other DNAsequences. Transcription enhancers or duplications of enhancers can beused to increase expression from a particular promoter. One may wish toobtain novel tissue-specific promoter sequences for use in accordancewith the present invention. To achieve this, one may first isolate CDNAclones from the tissue concerned and identify those clones which areexpressed specifically in that tissue, for example, using Northernblotting. Ideally, one would like to identify a gene that is not presentin a high copy number, but which gene product is relatively abundant inspecific tissues. The promoter and control elements of correspondinggenomic clones may then be localized using the techniques of molecularbiology known to those of skill in the art.

[0082] Expression of some genes in transgenic plants will occur onlyunder specified conditions. It is known that a large number of genesexist that respond to the environment. In some embodiments of thepresent invention expression of a DNA segment in a transgenic plant willoccur only in a certain time period during the development of the plant.Developmental timing is frequently correlated with tissue specific geneexpression.

[0083] As the DNA sequence inserted between the transcription initiationsite and the start of the coding sequence, i.e., the untranslated leadersequence, can influence gene expression, one can also employ aparticular leader sequence. Preferred leader sequence include thosewhich comprise sequences selected to direct optimum expression of theattached gene, i.e., to include a preferred consensus leader sequencewhich can increase or maintain mRNA stability and prevent inappropriateinitiation of translation (Joshi, 1987). Such sequences are known tothose of skill in the art. Sequences that are derived from genes thatare highly expressed in plants will be most preferred.

[0084] Additionally, expression cassettes can be constructed andemployed to target the gene product of the DNA segment to anintracellular compartment within plant cells or to direct a protein tothe extracellular environment. This can generally be achieved by joininga DNA sequence encoding a transit or signal peptide sequence to thecoding sequence of the DNA segment. Also, the DNA segment can bedirected to a particular organelle, such as the chloroplast rather thanto the cytoplasm.

[0085] Alternatively, the DNA fragment coding for the transit peptidemay be chemically synthesized either wholly or in part from the knownsequences of transit peptides such as those listed above. Thedescription of the optional sequences in the expression cassette, iscommonly owned, co-pending PCT/US/0027704, filed Oct. 6, 2000, entitled“Method of Introducing a Plurality of Genes into Plants,” incorporatedherein by reference.

[0086] Transformation

[0087] Transformation of cells from plants, e.g., trees, and thesubsequent production of transgenic plants using e.g.,Agrobacterium-mediated transformation procedures known in the art, andfurther described herein, is one example of a method for introducing aforeign gene into plants. Although, the method of the invention can beperformed by other modes of transformation, Agrobacterium-mediatedtransformation procedures are cited as examples, herein. For example,transgenic plants may be produced by the following steps: (i) culturingAgrobacterium in low-pH induction medium at low temperature andpreconditioning, i.e., coculturing bacteria with wounded tobacco leafextract in order to induce a high level of expression of theAgrobacterium vir genes whose products are involved in the T-DNAtransfer; (ii) coculturing desired plant tissue explants, includingzygotic and/or somatic embryo tissues derived from cultured explants,with the incited Agrobacterium; (iii) selecting transformed callustissue on a medium containing antibiotics; and (iv) converting theembryos into platelets.

[0088] Any non-tumorigenic A. tumefaciens strain harboring a disarmed Tiplasmid may be used in the method in accordance with the invention. AnyAgrobacterium system may be used. For example, Ti plasmid/binary vectorsystem or a cointegrative vector system with one Ti plasmid may be used.Also, any marker gene or polynucleotide conferring the ability to selecttransformed cells, callus, embryos or plants and any other gene, such asfor example a gene conferring resistance to a disease, or one improvinglignin content or structure or cellulose content, may also be used. Aperson of ordinary skill in the art can determine which markers andgenes are used depending on particular needs.

[0089] To increase the infectivity of the bacteria, Agrobacterium iscultured in low-pH induction medium, i.e., any bacterium culture mediawith a pH value adjusted to from 4.5 to 6.0, most preferably about 5.2,and at low temperature such as for example about 19-30° C., preferablyabout 21-26° C. The conditions of low-pH and low temperature are amongthe well-defined critical factors for inducing virulence activity inAgrobacterium (e.g., Altmorbe et al., 1989; Fullner et al., 1996;Fullner and Nester, 1996).

[0090] The bacteria is preconditioned by coculturing with woundedtobacco leaf extract (prepared according to methods known generally inthe art) to induce a high level of expression of the Agrobacterium virgenes. Prior to inoculation of plant somatic embryos, Agrobacteriumcells can be treated with a tobacco extract prepared from wounded leaftissues of tobacco plants grown in vitro. To achieve optimal stimulationof the expression of Agrobacterium vir genes by wound-inducedmetabolites and other cellular factors, tobacco leaves can be woundedand pre-cultured overnight. Culturing of bacteria in low pH medium andat low temperature can be used to further enhance the bacteria vir geneexpression and infectivity. Preconditioning with tobacco extract and thevir genes involved in the T-DNA transfer process are generally known inthe art.

[0091] Agrobacterium treated as described above is then cocultured witha plant tissue explant, such as for example, zygotic and/or somaticembryo tissue. Non-zygotic (i.e., somatic) or zygotic tissues can beused. Any plant tissue may be used as a source of explants. For example,cotyledons from seeds, young leaf tissue, root tissues, parts of stemsincluding nodal explants, and tissues from primary somatic embryos suchas the root axis may be used. Generally, young tissues are a preferredsource of explants.

[0092] The above-described transformation and regeneration protocol isreadily adaptable to other plant species. Other published transformationand regeneration protocols for plant species include Danekar et al,1987; McGranahan et al, 1988; McGranahan et al., 1990; Chen, Ph.D.Thesis, 1991; Sullivan et al, 1993; Huang et al., 1991; Wilde et al.,1992; Minocha et al., 1986; Parsons et al., 1986; Fillatti et al., 1987;Pythoud et al., 1987; De Block, 1990; Brasileiro et al., 1991;Brasileiro et al., 1992; Howe et al., 1991; Klopfenstein et al., 1991;Leple et al., 1992; and Nilsson et al., 1992.

[0093] Characterization

[0094] To confirm the presence of the DNA segment(s) or “transgene(s)”in the regenerated plants, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting andPCR; “biochemical” assays, such as detecting the presence of a proteinproduct, e.g., by immunological means (ELISAs and Western blots) or byenzymatic function; plant part assays, such as leaf or root assays; andalso, by analyzing the phenotype of the whole regenerated plant.

[0095] 1. DNA Integration, RNA Expression and Inheritance

[0096] Genomic DNA may be isolated from callus cell lines or any plantparts to determine the presence of the DNA segment through the use oftechniques well known to those skilled in the art. Note that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell.

[0097] The presence of DNA elements introduced through the methods ofthis invention may be determined by polymerase chain reaction (PCR).Using this technique, discreet fragments of DNA are amplified anddetected by gel electrophoresis. This type of analysis permits one todetermine whether a DNA segment is present in a stable transformant, butdoes not prove integration of the introduced DNA segment into the hostcell genome. In addition, it is not possible using PCR techniques todetermine whether transformants have exogenous genes introduced intodifferent sites in the genome, i.e., whether transformants are ofindependent origin. It is contemplated that using PCR techniques itwould be possible to clone fragments of the host genomic DNA adjacent toan introduced DNA segment.

[0098] Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique, specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition, it is possible through Southernhybridization to demonstrate the presence of introduced DNA segments inhigh molecular weight DNA, i.e., confirm that the introduced DNA segmenthas been integrated into the host cell genome. The technique of Southernhybridization provides information that is obtained using PCR, e.g., thepresence of a DNA segment, but also demonstrates integration into thegenome and characterizes each individual transformant.

[0099] It is contemplated that by using the techniques of dot or slotblot hybridization which are modifications of Southern hybridizationtechniques, one could obtain the same information that is derived fromPCR, e.g., the presence of a DNA segment.

[0100] Both PCR and Southern hybridization techniques can be used todemonstrate transmission of a DNA segment to progeny. In most instancesthe characteristic Southern hybridization pattern for a giventransformant will segregate in progeny as one or more Mendelian genes(Spencer et al., 1992; Laursen et al.,1994) indicating stableinheritance of the gene.

[0101] Whereas DNA analysis techniques may be conducted using DNAisolated from any part of a plant, RNA may only be expressed inparticular cells or tissue types, and hence, it will be necessary toprepare RNA for analysis from these tissues. PCR techniques may also beused for detection and quantitation of RNA produced from introduced DNAsegments. In this application of PCR, it is first necessary to reversetranscribe RNA into DNA, using enzymes such as reverse transcriptase,and then through the use of conventional PCR techniques amplify the DNA.In most instances, PCR techniques, while useful, will not demonstrateintegrity of the RNA product. Further information about the nature ofthe RNA product may be obtained by Northern blotting. This techniquewill demonstrate the presence of an RNA species and give informationabout the integrity of that RNA. The presence or absence of an RNAspecies can also be determined using dot or slot blot Northernhybridizations. These techniques are modifications of Northern blottingand demonstrate only the presence or absence of an RNA species.

[0102] 2. Gene Expression

[0103] While Southern blotting and PCR may be used to detect the DNAsegment in question, they do not provide information as to whether theDNA segment is being expressed. Expression may be evaluated byspecifically identifying the protein products of the introduced DNAsegments or evaluating the phenotypic changes brought about by theirexpression.

[0104] Assays for the production and identification of specific proteinsmay make use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focussing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins also offer opportunities foruse of specific antibodies to detect their presence in formats such asan ELISA assay. Combinations of approaches may be employed with evengreater specificity such as western blotting in which antibodies areused to locate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

[0105] Assay procedures may also be used to identify the expression ofproteins by their functionality, especially the ability of enzymes tocatalyze specific chemical reactions involving specific substrates andproducts. These reactions may be followed by providing and quantifyingthe loss of substrates or the generation of products of the reactions byphysical or chemical procedures. Examples are as varied as the enzyme tobe analyzed and may include assays for PAT enzymatic activity byfollowing production of radiolabelled acetylated phosphinothricin fromphosphinothricin.

[0106] Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of DNA segmentsencoding enzymes or storage proteins which change amino acid compositionand may be detected by amino acid analysis, or by enzymes which changestarch quantity which may be analyzed by near infrared reflectancespectrometry. Morphological changes may include greater stature orthicker stalks. Most often changes in response of plants or plant partsto imposed treatments are evaluated under carefully controlledconditions termed bioassays.

[0107] The invention will be further described by the followingnon-limiting examples.

EXAMPLE 1 Preparation of Transgenic Aspen

[0108] Construction of binary vectors

[0109] pBKPpt_(4CL) Pt4CL1-a: Aspen 4CL1 xylem specific promoter(Ppt_(4CL,) 1.1 kb, GenBank AF041051) was prepared and linked to aspen4CL1 cDNA (Pt4CL1, GenBank AF041049) which was orientated in theantisense direction. Then the cassette containing aspen 4CL1 promoterand antisense aspen 4CL1 cDNA was positioned in a plant transformationbinary vector, as shown in FIG. 1. (pBKPpt_(4CL) Pt4CL1-a construct)

[0110] pBKPpt_(4cl) PtCAld5H-s: From pBKPpt_(4CL) Pt4CL-a construct, theantisense Pt4CL1 was replaced with PtCAld5H cDNA in a sense orientation,yielding a pBKPpt_(4CL) PtCAld5H-s transformation binary construct, asshown in FIG. 2.

[0111] Also, Example 1 of PCT application PCT/US/0027704, filed Oct. 6,2000, entitled “Method of Introducing a Plurality of Genes into Plants,”incorporated herein by reference, describes a number of other geneconstructs for preparing transgenic plants. The plants are transformedwith a genes from the phenylpropanoid pathway (i.e., 4CL, AEOMT, CoAOMT,and CAld5H) using an operably linked to either a homologous or aheterologous and either a constitutive or tissue-specific promoter

[0112] Incorporation of binary vector into Agrobacterium

[0113] According to the protocol described in Tsai et al. (1994, PlantCell Reports, 14:94-97) Agrobacterium C58/pMP90 strain was grown in LBwith selection of gentamicin at 28° C. overnight. Cells were harvestedby centrifugation at 10,000 rpm for 10 minutes at 4° C. The cell pelletwas washed with 0.5 volume of ice-cold 20 mM CaCl₂, and centrifugedagain. The cells were then resuspended in 0.1 volume of ice-cold 20 mMCaCl₂ in a sample tube. About 1 μg of binary vector DNA was added to 200μL of the cell suspension and mixed by pipetting. The sample tube waschilled in liquid N₂ for 5 minutes and thawed at 37° C. in a water bathfor 5 minutes. One mL of LB medium was added and the mixture wasincubated at 28° C. for 3 hours with gentle shaking. Twenty μL of thecells were spread onto a LB plate containing 25 μg/mL gentamicin and 50μg/mL kanamycin and incubated at 28° C. for 2 days. PCR (amplificationconditions, cycling parameters and primers are described below) was usedto verify the presence of DNA from the vector in the transformedcolonies.

[0114] Simultaneous transformation of Aspen with multiple genes viaengineered Agrobacterium strains

[0115] For simultaneous transformation of multiple genes, pBKPpt_(4cl)Pt4CL-a and pBKPpt_(4cl) PtCal5H Agrobacterium clones were cultured inLB medium at 28° C. overnight separately. The Agrobacterium strains weresubcultured individually by a 100-fold dilution into 50 mL of LB (pH5.4) containing 50 μg/mL kanamycin, 25 μg/mL gentamycin and 20 μMacetosyringone (in DMSO), and grown overnight at 28° C. with shaking. Anequal volume of the same density of individually cultured Agrobacteriumstrains was then mixed. Leaves excised from sterile tobacco plants werecut into pieces with a size of about 5 mm² and the leaf discs were thenimmersed in the Agrobacterium mixture for 5 minutes.

[0116] After removing excess Agrobacterium cells, the treated leaf discswere placed on callus induction medium (WPM:Woody Plant Medium, BA:6-benzyladenine +2,4-D: 2,4-dichlorophenoxyacetic acid; Tsai et al.1994, Plant Cell Reports, 14:94-97) and cultured for 2 days. Then, thepre-cultured leaf discs were rinsed with sterile water several times toremove the Agrobacterium cells and washed in 1 mg/mL claforan and 1mg/mL ticarcillin with shaking for 3 hours to kill Agrobacterium. Afterbriefly blot-drying, the pre-cultured and washed leaf discs werecultured on callus induction medium containing 50 μg/mL kanamycin and300 μg/mL claforan for selection of transformed cells. After 2 to 3subcultures (10 days/subculture), the calli grown on the leaf discs wereexcised and transferred onto shoot induction medium (WPM+TDZ:N-phenyl-N′-1,2,3-thiadiazol-5-yl-urea) containing 50 μg/ml kanamycinand 300 μg/ml claforan for regenerating shoots. After shoots were grownto about 0.5 cm high, they excised and planted to rooting media (WPMwith kanamycin and claforan). Whole plants about 7 cm high weretransplanted into soil and maintained in a greenhouse for subsequentmolecular characterization.

[0117] Genomic DNA isolation

[0118] Genomic DNA was isolated according to Hu et al. (1998). About 100mg of young leaves were collected from each plant growing in thegreenhouse and ground in liquid N₂ to fine powder for DNA isolationusing QIAGEN plant DNA isolation kit (Valencia, Calif.). Specifically,the powdered tissue was added to extract buffer containing 2%hexadecyltrimethylammonium bromide (CTAB), 100 mM Tris-HC1, pH 8.0, 20mM EDTA, 1.4 M NaCl and 30 mM β-mercaptoethanol at 5 mL/g tissue. Theextraction mixture was incubated in a tube at 60° C. for 1 hour withoccasional shaking. One volume of chloroform-isoamyl alcohol (24:1) wasadded and mixed gently. The two phases were separated by centrifugationat 10,000×g for 10 minutes. The aqueous phase was transferred to a newtube and extracted with chloroform in the presence of 1% CTAB and 0.7 MNaCl. The DNA was precipitated by addition of ⅔ volume of isopropanol(−20° C.) and kept at −20° C. for 20 minutes. Following thecentrifugation at 10,000×g for 10 minutes, the pelleted DNA was washedwith 70% ethanol-10 mM ammonia acetate. Then the pellet was dissolved in2 mL TE buffer (10 mM Tris-HC1/0.1 mM EDTA, pH 8) and treated with 2 μgRNase A at 37° C. for 20 minutes. The DNA was precipitated by additionof 2 mL of 5 M ammonia acetate and 10 mL of 95% ethanol at −20° C. for20 minutes. After centrifugation, the pellet was washed with 70%ethanol. After a brief drying, genomic DNA was dissolved in TE buffer.

[0119] PCR verification of foreign gene insertion in host plant genome

[0120] PCR was used to verify the integration of the gene constructs inthe genome of transgenic plants. Two specific primers were synthesizedfor each construct and used to PCR-amplify the corresponding constructin genome of transgenic Aspen. For the PBKPpt_(4CL) Pt4CL1-a construct,two specific primers were synthesized that amplify a 4CL cDNA fragment.Pt4CL1 promoter sense primer (5′CAGGAATGCTCTGCACTCTG3′) (SEQ ID NO:11)and Pt4CL1 sense primer (5′ATGAATCCACAAGAATTCAT3′) (SEQ ID NO:12). atthe translation start region. Primers for PCR verification ofpBKPpt_(4CL) PtCald5H-s construct are Pt4CL1 promoter sense primer(5′CAGGAATGCTCTGCACTCTG3′) (SEQ ID NO:13) and PtCald5H antisense primer(5′TTAGAGAGGACAGAGCACACG3′) (SEQ ID NO:14) at translation stop region.

[0121] The PCR reaction mixture contained 100 ng genomic DNA oftransformed aspen, and 0.2 μM of each primer, 100 μM of eachdeoxyribonucleotide triphosphate, 1×PCR buffer and 2.5 Units of Taq DNApolymerase (Promega Madison, Wis.) in a total volume of 50 μL. Thecycling parameters were as follows: 94° C. for 1 minute, 56° C. for 1minute (for 4CL and CAld5H or can vary between cDNA templates used)according to different gene checked) and 72° C. for 2 minute, for 40cycles, with 5 minutes at 72° C. extension. The PCR products wereelectrophoresized on a 1% agarose gel.

EXAMPLE 2 Preparation of other transgenic plants

[0122] It is important to recognize that there is a substantialpercentage of sequence homology among the plant genes involved in thelignin biosynthetic pathway, discussed herein. This substantial sequencehomology allows the method in accordance with the invention disclosedherein to be applicable to all plants that possess the requisite genesinvolved in the lignin biosynthetic pathway. To demonstrate thesubstantial sequence homology among plant genes, the percentage sequencehomology is set forth in tabular form, for example, CAld5H genes (Table1), AldOMT genes (Table 2), CAD genes (Table 3), and 4CL genes (See FIG.12). Therefore, it is possible to alter lignin monomer composition,increase S/G lignin ratio, and increase cellulose content in all plantsby using the method in accordance with the invention, described herein.TABLE 1 Protein sequence homology (%) of plant Coniferyl Aldehyde5-hydroxylase (CAld5H) from 1) Aspen; 2) Poplar, AJ010324; 3) Sweetgum,AF139532; 4) Arabidopsis (Ferulic Acid 5-hydroxylase, F5H) 1 2 3 4 1 299 3 84 84 4 81 83 83

[0123] TABLE 2 Protein sequence homology (%) of plant AldOMTs from 1)Aspen, X62096; 2) Poplar, M73431; 3) Almond, X83217; 4) Strawberry,AF220491; 5) Alfalfa, M63853; 6) Eucalyptus, X74814; 7) Clarkia breweri,AF006009; 8) Sweetgum, AF139533; 9) Arabidopsis, U70424; 10) Tobacco,X74452; 11) Vitis vinifera, AF239740 1 2 3 4 5 6 7 8 9 10 11 1 2 99 3 9292 4 91 90 94 5 90 90 89 89 6 89 89 89 87 87 7 88 88 89 88 87 90 8 88 8788 87 86 85 83 9 84 84 85 86 82 82 82 83 10 83 83 83 82 81 82 80 83 7711 80 80 78 77 78 77 78 80 76 77

[0124] TABLE 3 Protein sequence homology (%) of plant CADs from 1)Aspen, AF217957; 2) Cottonwood, Z19568 and 3) Udo, D13991; 4) Tobacco,X62343; 5) Tobacco, X62344; 6) Eucalyptus, AF038561; 7) Eucalyptus,X65631; 8) Lucerne, AF083332; 9) Lucerne, Z19573; 10) Maize, AJ005702;11) Maize, Y13733; 12) Sugarcane, AJ231135; 13) Radiata pine, U62394;14) Loblolly pine, Z37992; 15) Loblolly pine, Z37991; 16) Norway spruce,X72675. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 97 3 85 84 4 82 82 845 80 80 81 94 6 81 81 82 80 78 7 81 80 81 80 78 80 8 79 79 80 80 79 7979 9 79 80 80 79 78 78 79 99 10 78 77 79 76 74 76 77 73 73 11 78 78 7977 74 76 76 73 72 99 12 77 76 78 74 73 75 74 73 73 95 96 13 70 71 69 7070 69 68 67 68 67 68 68 14 69 70 69 69 69 69 68 68 68 67 67 67 99 15 6970 68 69 69 68 68 67 67 67 67 67 99 95 16 69 69 70 70 69 68 68 68 67 6969 67 95 95 94

[0125] To further demonstrate the versatility of this invention intransferring a variety of foreign genes and the applicability of thisinvention to plants other than the herbaceous species, different binaryvectors were constructed and transferred into aspen (Populustremuloides) tree. Two binary vectors, each containing a cDNA sequenceand a neomycin phosphotransferase (NPT II) cDNA encoding kanamycinresistance, were constructed. Each vector was then individuallymobilized into Agrobacterium strain C58 to create two isolated(engineered) Agrobacterium strains. It should be noted that about 50transgenic tobacco plants were generated by the same technique harboring4 different sets of foreign genes, as described in the PCT applicationPCTUS0027704 filed Oct. 6, 2000, entitled “Method of Introducing aPlurality of Genes into Plants,” incorporated herein by reference.

[0126] Table 4 summarizes the numerical results from simultaneousmanipulating xylem-specific expression of 4CL and CAld5H in transgenicaspen. After DNA constructs were incorporated into plant cells byAgrobacterium mediated transformation, as set forth by the method inaccordance with the invention and after PCR confirmation of transgeneintegration, 14 positive transgenic trees were randomly selected,representing three different trangenic groups, i.e., Groups I, II andIII. Group I (plant #21, 22, 23, 25, and 37) consists of those with theintegration of only antisense Pt4CL1 cDNA (Table 4). Group II plants (#32, 84, 93, and 94) harbored only sense PtCAld5H cDNA, whereas Group IIIplants (#71, 72, 74, and 141) contained both antisense Pt4CL1 and sensePtCAld5H transgenes. These transgenic trees were then further analyzedfor their lignin and cellulose contents and lignin S/G ratio (Table 4).It is clear that, when compared with the control, untransformed aspen,transgenic plants (#21, 22, 23, 25, and 37) engineered for thesuppression of 4CL gene with antisense Pt4CL1 transgene had drasticreductions in their lignin content, with significant increases in theircellulose content. Transgenic plants (#32, 84, 93, 94, and 108)engineered for the overexpression of CAld5H with sense PtCAld5Htransgene had pronounced increases in their S/G ratio, but their ligninand cellulose contents remained essentially unaffected. When engineeredfor the simultaneous suppression of 4CL gene and overexpression ofCAld5H gene, transgenic plants (#71, 72, 74, and 141) all exhibited lowlignin content, high S/G ratio and elevated cellulose quantity. Insummary, these results show that multiple genes carried by individualAgrobacterium strains can be integrated simultaneously into the plantgenome.

[0127] Moreover, it was demonstrated as shown herein below, thattransgenic plants with a nearly 30% increase in cellulose content andover 50% lignin quantity reduction, accompanied with a significantaugmentation of the S/G ratio, can be easily produced. It is conceivablethat more genes can also be efficiently transferred at one time. Onlyone suitable marker gene is required for this system, although a numberof marker genes can also be employed. TABLE 4 Simultaneous manipulatingxylem-specific expression of 4CL and CAld5H in transgenic aspen. Plant #Control 21 22 23 25 37 32 84 93 94 108 71 72 74 141 Gene 4CL-a Y Y Y Y YY Y Y Y integrated CAld5H-s Y Y Y Y Y Y Y Y Y Lignin content (%) 22.416.0 15.3 14.4 13.1 14.9 22.4 21.6 21.1 20.7 19.7 13.2 13.7 12.4 10.7Lignin S/G ratio 2.2 2.1 2.0 2.2 2.3 2.1 4.8 4.0 5.5 4.9 3.0 3.3 3.6 3.42.7 Cellulose content (%) 41.4 43.1 ND ND 47.3 ND 40.0 ND 44.7 ND ND ND49.2 ND 53.3

EXAMPLE 3 Production of commercially desirable agronomic traits intransformed plants.

[0128] The following genetic transformations illustrate the productionof commercially desirable agronomic traits in plants.

[0129] Gymnosperms

[0130] A. To produce syringyl-enriched lignin in gymnosperm plants,gymnosperm plants are genetically transformed with SAD, CAld5H, andAldOMT genes in the sense orientation driven by any appropriate promoterand via any appropriate genetic transformation system allows. Thesethree genes can be transferred into the host plant either simultaneously(in one or individual constructs) or sequentially (in individualconstructs) in any order.

[0131] B. To produce decreased lignin content, increasedsyringyl/guaiacyl (S/G) lignin ratio and increased cellulose quantity ingymnosperm plants, gymnosperm plants are genetically transformed withSAD, CAld5H and AldOMT genes in the sense orientation and 4CL gene ineither sense or antisense orientation driven by any appropriate promoterand via any appropriate genetic transformation system. These four genescan be transferred into the host plant either simultaneously (in one orindividual constructs) or sequentially (in individual constructs) in anyorder.

[0132] C. To produce decreased lignin content, increasedsyringyl/guaiacyl (S/G) lignin ratio and increased cellulose quantity ingymnosperm plants, gymnosperm plants are genetically transformed withSAD, CAld5H and AldOMT genes in the sense orientation and 4CL and CADgenes in either sense or antisense orientation driven by any appropriatepromoter and via any appropriate genetic transformation system. Thesefive genes can be transferred into the host plant either simultaneously(in one or individual constructs) or sequentially (in individualconstructs) in any order.

[0133] D. To produce increased lignin content in gymnosperm plants,gymnosperm plants are genetically transformed with 4CL gene in the senseorientation driven by any appropriate promoter and via any appropriategenetic transformation system.

[0134] E. To produce increased lignin content and increasedsyringyl/guaiacyl (S/G) lignin ratio in gymnosperm plants, gymnospermplants are genetically transformed with SAD, CAld5H, AldOMT, and 4CLgenes in the sense orientation driven by any appropriate promoter andvia any appropriate genetic transformation system. These four genes canbe transferred into the host plant either simultaneously (in one orindividual constructs) or sequentially (in individual constructs) in anyorder.

[0135] F. To produce increased lignin content, increasedsyringyl/guaiacyl (S/G) lignin ratio in gymnosperm plants, gymnospermplants are genetically transformed with SAD, CAld5H, AldOMT, and 4CLgenes in the sense orientation and CAD gene in the antisense orientationdriven by any appropriate promoter and via any appropriate genetictransformation system. These four genes can be transferred into the hostplant either simultaneously (in one or individual constructs) orsequentially (in individual constructs) in any order.

[0136] Angiosperms

[0137] A. To produce increased S/G lignin ratio in angiosperm plants,angiosperm plants are genetically transformed with either CAld5H,AldOMT, or SAD genes in sense orientation driven by any appropriatepromoter and via any appropriate genetic transformation system. Thesethree genes can be transferred into the host plant either simultaneously(in one or individual constructs) or sequentially (in individualconstructs) in any order.

[0138] B. To produce decreased lignin content, increased S/G ligninratio and increased cellulose quantity in angiosperm plants, angiospermplants are genetically transformed with either SAD, CAld5H, or AldOMTgenes in the sense orientation and 4CL gene in the sense or antisenseorientation driven by any appropriate promoter and via any appropriategenetic transformation system. These four genes can be transferred intothe host plant either simultaneously (in one or individual constructs)or sequentially (in individual constructs) in any order.

[0139] C. To produce decreased lignin content, increased S/G ligninratio and increased cellulose quantity in angiosperm plants, angiospermplants are genetically transformed with either SAD, CAld5H, or AldOMTgenes in the sense orientation and 4CL and CAD genes in the sense orantisense orientation driven by any appropriate promoter and via anyappropriate genetic transformation system. These five genes can betransferred into the host plant either simultaneously (in one orindividual constructs) or sequentially (in individual constructs) in anyorder.

[0140] D. To produce increased lignin content in angiosperm plants,angiosperm plants are genetically transformed with 4CL gene in the senseorientation driven by any appropriate promoter and via any appropriategenetic transformation system.

[0141] E. To produce increased lignin content and increased S/G ratio inangiosperm plants, angiosperm plants are genetically transformed with4CL in the sense orientation and either SAD, CAld5H, or AldOMT genesalso in the sense orientation driven by any appropriate promoter and viaany appropriate genetic transformation system. These four genes can betransferred into the host plant either simultaneously (in one orindividual constructs) or sequentially (in individual constructs) in anyorder.

[0142] F. To produce increased lignin content and increased S/G ratio inangiosperm plants, angiosperm plants are genetically transformed with4CL in the sense orientation and either SAD, CAld5H, or AldOMT genesalso in the sense orientation and CAD in the antisense orientationdriven by any appropriate promoter and via any appropriate genetictransformation system. These four genes can be transferred into the hostplant either simultaneously (in one or individual constructs) orsequentially (in individual constructs) in any order.

[0143] All publications, patents and patent applications cited hereinare incorporated herein by reference. While in the foregoingspecification, this invention has been described in relation to certainpreferred embodiments thereof, and many details have been set forth forpurposes of illustration, it will be apparent to those skilled in theart that the invention is susceptible to additional embodiments and thatcertain of the details herein may be varied considerably withoutdeparting from the basic principles of the invention. Accordingly, it isintended that the present invention be solely limited by the broadestinterpretation that can be accorded the appended claims.

REFERENCES

[0144] Bugos et al., 1991, Plant Mol. Biol. 17:203.

[0145] Chang, H. M., and Sarkanen, K. V., 1973, Tappi 56:132.

[0146] Chiang, V. L., and Funaoka, M., 1990, Holzforschung 44:309.

[0147] Hu et al., 1999, Nature Biotech. 17:808.

[0148] Sarkanen, K. V., and Ludwig, C. H., eds (Wiley-Interscience, NewYork), 639.

[0149] Tsai et al., 1994, Plant Cell Report 14:94.

[0150] Boudet et al., 1995, New Phytol. 129:203.

[0151] Ibrahim, 1997, Trends Plant Sci. 2:249.

[0152] Li et al., 1997, Proc. Natl. Acad. Sci. USA 94:5461.

[0153] Joshi and Chiang, 1998, Plant Mol. Biol. 37:663.

[0154] Brasileiro et al., 1991, Plant Mol. Bio. 17:441.

[0155] Brasileiro et al., 1992, Transgenic Res. 1:133.

[0156] Chen et al., 1998, Nature Biotechnology 16, 11:1060.

[0157] Chen, Ph.D. Thesis, 1991, North Carolina State University,Raleigh, N.C.

[0158] Chen et al.,1999, Planta 207:597.

[0159] Christou, 1996, Bio/Technology 10:667.

[0160] Chandler et al., 1989.

[0161] Danekar et al., 1987, Bio/Technology 5:587.

[0162] De Block, 1990, Plant Physiol. 93:1110.

[0163] Ebinuma et al., 1997, Proceedings of the National Academic ofSciences 94:2117.

[0164] Ebert et al. 1987.

[0165] Fillatti et al., 1987, Mol. Gen. Genet. 206:192.

[0166] Freudenberg, 1965.

[0167] Horsch et al., 1985, Science 227:1229.

[0168] Howe et al., 1991, Woody Plant Biotech. Plenum Press, New York,283.

[0169] Huang et al., 1991, In Vitro Cell Dev. Bio. 4:201.

[0170] Hudspeth et al., 1989, Plant Mol. Biol., 12:579.

[0171] Hu et al., 1998, Proc. Natl. Acad. Sci. USA 95:5407.

[0172] Hu et al., 1999, Nat. Biotechnol. 17:808.

[0173] Humphreys et al., 1999, Proc. Nat. Acad. Sci. USA 96:10045.

[0174] Jornvall et al., 1987, Eur. J. Biochem. 167:195.

[0175] Jefferson et al.,1987.

[0176] Klopfenstein et al., 1991, Can. J For. Res. 21:1321.

[0177] Lawton et al., 1987, Plant Mol. Biol. 9:31F.

[0178] Buxton and Roussel, 1988, Crop. Sci. 28,:553.

[0179] Jung and Vogel, 1986, J. Anim., Sci. 62:1703.

[0180] Leple et al., 1992, Plant Cell Reports 11:137.

[0181] Li et al., 1997, Proc. Natl. Acad. Sci. USA, 94:5461.

[0182] Li et al., 2001, Plant Cell, 13:1567.

[0183] Li et al, 1997, Proc. Natl. Acad. Sci. USA 94:5431.

[0184] Li et al., 1999, Plant Mol. Biol. 40:555.

[0185] Li et al., 2000, J. Biol. Chem. 275:6537.

[0186] MacKay et al., 1995, Mol. Gen. Genet. 247:537.

[0187] MacKay et al., 1997.

[0188] McGranahan et al., 1988, Bio/Technology 6:800.

[0189] McGranahan et al., 1990, Plant Cell Reports 8:512.

[0190] Minocha et al., 1986, Proc. TAPPI Research and DevelopmentConference, TAPPI Press, Atlanta, 89.

[0191] Nelson et al. 1996, Pharmacogenetics 6: 1.

[0192] Odell et al., 1985, Nature 313:810.

[0193] Osakabe et al., 1999, Proc. Nati. Acad. Sci. USA 96:8955.

[0194] Parsons et al., 1986, Bio/Technology 4:533.

[0195] Pythoud et al., 1987, Bio/Technology 5:1323.

[0196] Sambrook et al., 2 ^(nd) ed. 1982.

[0197] Sullivan et al., 1993, Plant Cell Reports 12:303.

[0198] Sarkanen, K. V., and Hergert, H. L., 1971, Lignins: Occurrence,Formation, Structure and Reaction, K. V. Sarkanen and C. H. Ludwig, eds(New York: Wiley-Interscience), 43.

[0199] Trotter, P. C., 1990, Tech. Assoc. Pulp Paper Ind. J. 73:198.

[0200] Tsai et al., 1998, Plant Physiol. 117:101.

[0201] Tsai et al., Plant Cell Reports 14:94.

[0202] Tricoli et al., 1995.

[0203] Walker et al., 1987, PNAS USA 84:6624.

[0204] Wang et al., 1992, Mol. Cell. Biol. 12:3399.

[0205] Wu et al., 2000, Plant J. 22:495.

[0206] Yang et al., 1990, PNAS USA 87:4144.

[0207] Yamazaki et al., 1993, J. Biochem. 114:652.

[0208] Zhang, X.-H., and Chiang, V. L., 1997, Plant Physiol. 113:65.

1 14 1 1446 DNA aspen populus tremuloides misc_feature SAD 1 tttttttttttttcctagcc ttccttctcg acgatatttc tctatctgaa gcaagcacca 60 tgtccaagtcaccagaagaa gaacaccctg tgaaggcctt cgggtgggct gctagggatc 120 aatctggtcatctttctccc ttcaacttct ccaggagggc aactggtgaa gaggatgtga 180 ggttcaaggtgctgtactgc gggatatgcc attctgacct tcacagtatc aagaatgact 240 ggggcttctccatgtaccct ttggttcctg ggcatgaaat tgtgggggaa gtgacagaag 300 ttgggagcaaggtgaaaaag gttaatgtgg gagacaaagt gggcgtggga tgcttggttg 360 gtgcatgtcactcctgtgag agttgtgcca atgatcttga aaattactgt ccaaaaatga 420 tcctgacatacgcctccatc taccatgacg gaaccatcac ttacggtggc tactcagatc 480 acatggtcgctaacgaacgc tacatcattc gattccccga taacatgccg cttgacggtg 540 gcgctcctctcctttgtgcc gggattacag tgtatagtcc cttgaaatat tttggactag 600 atgaacccggtaagcatatc ggtatcgttg gcttaggtgg acttggtcac gtggctgtca 660 aatttgccaaggcctttgga tctaaagtga cagtaattag tacctcccct tccaagaagg 720 aggaggctttgaagaacttc ggtgcagact catttttggt tagtcgtgac caagagcaaa 780 tgcaggctgccgcaggaaca ttagatggca tcatcgatac agtttctgca gttcaccccc 840 ttttgccattgtttggactg ttgaagtctc acgggaagct tatcttggtg ggtgcaccgg 900 aaaagcctcttgagctacct gccttttctt tgattgctgg aaggaagata gttgccggga 960 gtggtattggaggcatgaag gagacacaag agatgattga ttttgcagca aaacacaaca 1020 tcacagcagatatcgaagtt atttcaacgg actatcttaa tacggcgata gaacgtttgg 1080 ctaaaaacgatgtcagatac cgattcgtca ttgacgttgg caatactttg gcagctacga 1140 agccctaaggagaagatccc atgttctcga accctttata aaatctgata acatgtgttg 1200 atttcatgaataaatagatt atctttggga tttttcttta ataaacgaag tgttctcgaa 1260 aacttaacatcggcaatacc ctggcagcta cgagaaacgc tttagaattg tttgtaagtt 1320 tgtttcattagggtgatacc atgctctcga gtcctttgta agatccattt atagttgcgt 1380 gaatgctatgaacaaataat atgtttgcgg cttctcttca aaaaaaaaaa aaaaaaaaaa 1440 aaaaaa 14462 362 PRT aspen populus tremuloides 2 Met Ser Lys Ser Pro Glu Glu GluHis Pro Val Lys Ala Phe Gly Trp 1 5 10 15 Ala Ala Arg Asp Gln Ser GlyHis Leu Ser Pro Phe Asn Phe Ser Arg 20 25 30 Arg Ala Thr Gly Glu Glu AspVal Arg Phe Lys Val Leu Tyr Cys Gly 35 40 45 Ile Cys His Ser Asp Leu HisSer Ile Lys Asn Asp Trp Gly Phe Ser 50 55 60 Met Tyr Pro Leu Val Pro GlyHis Glu Ile Val Gly Glu Val Thr Glu 65 70 75 80 Val Gly Ser Lys Val LysLys Val Asn Val Gly Asp Lys Val Gly Val 85 90 95 Gly Cys Leu Val Gly AlaCys His Ser Cys Glu Ser Cys Ala Asn Asp 100 105 110 Leu Glu Asn Tyr CysPro Lys Met Ile Leu Thr Tyr Ala Ser Ile Tyr 115 120 125 His Asp Gly ThrIle Thr Tyr Gly Gly Tyr Ser Asp His Met Val Ala 130 135 140 Asn Glu ArgTyr Ile Ile Arg Phe Pro Asp Asn Met Pro Leu Asp Gly 145 150 155 160 GlyAla Pro Leu Leu Cys Ala Gly Ile Thr Val Tyr Ser Pro Leu Lys 165 170 175Tyr Phe Gly Leu Asp Glu Pro Gly Lys His Ile Gly Ile Val Gly Leu 180 185190 Gly Gly Leu Gly His Val Ala Val Lys Phe Ala Lys Ala Phe Gly Ser 195200 205 Lys Val Thr Val Ile Ser Thr Ser Pro Ser Lys Lys Glu Glu Ala Leu210 215 220 Lys Asn Phe Gly Ala Asp Ser Phe Leu Val Ser Arg Asp Gln GluGln 225 230 235 240 Met Gln Ala Ala Ala Gly Thr Leu Asp Gly Ile Ile AspThr Val Ser 245 250 255 Ala Val His Pro Leu Leu Pro Leu Phe Gly Leu LeuLys Ser His Gly 260 265 270 Lys Leu Ile Leu Val Gly Ala Pro Glu Lys ProLeu Glu Leu Pro Ala 275 280 285 Phe Ser Leu Ile Ala Gly Arg Lys Ile ValAla Gly Ser Gly Ile Gly 290 295 300 Gly Met Lys Glu Thr Gln Glu Met IleAsp Phe Ala Ala Lys His Asn 305 310 315 320 Ile Thr Ala Asp Ile Glu ValIle Ser Thr Asp Tyr Leu Asn Thr Ala 325 330 335 Ile Glu Arg Leu Ala LysAsn Asp Val Arg Tyr Arg Phe Val Ile Asp 340 345 350 Val Gly Asn Thr LeuAla Ala Thr Lys Pro 355 360 3 1764 DNA aspen populus tremuloidesmisc_feature CAld5H 3 taaagtcttg tggattacac aaaatacaga ctgaaaacatccataggcac caacacataa 60 accatccatg gattctcttg tccaatcttt gcaagcttcacccatgtctc tcttcttgat 120 cgttatctct tcactcttct tcttcggtct cctctctcgccttcgccgaa gattgccata 180 tccaccaggg cctaaagggt tgccacttgt aggtagcatgcacatgatgg accaaataac 240 tcaccgtggg ttagctaaac tagctaagca atatggtgggctctttcata tgcgcatggg 300 gtacttgcat atggtcactg tttcatctcc tgaaatagctcgccaagttc tgcaggtcca 360 ggacaacatt ttctccaaca gaccagccaa catagccataagttacttaa cctatgatcg 420 tgcagatatg gcctttgccc actacggtcc tttctggcgacagatgcgta agctctgcgt 480 catgaagctt tttagccgga aaagggctga atcatgggagtctgtgagag atgaggtgga 540 ctcaatgctt aagacagttg aagccaatat aggcaagcctgtgaatcttg gggaattgat 600 ttttacgttg accatgaaca tcacttacag agcagctttcggggctaaaa atgaaggaca 660 ggatgagttc atcaagattt tgcaggagtt ctctaagctttttggagcat tcaacatgtc 720 tgatttcatt ccctggctgg gctggattga cccccaagggctcagcgcta gacttgtcaa 780 ggctcgcaag gctcttgata gattcatcga ctctatcatcgatgatcata tccagaaaag 840 aaaacagaat aagttctctg aagatgctga aaccgatatggtcgatgaca tgctagcctt 900 ttatggtgaa gaagcaagga aagtagatga atcagatgatttacaaaaag ccatcagcct 960 tactaaagac aacatcaaag ccataatcat ggatgtgatgtttggtggga cagagacggt 1020 ggcgtcggca atagagtggg tcatggcgga gctaatgaagagtccagagg atcaaaaaag 1080 agtccagcaa gagctcgcag aggtggtggg tttagagcggcgcgtggagg aaagtgatat 1140 tgacaaactt acgttcttga aatgcgccct caaagaaaccttaaggatgc acccaccaat 1200 cccacttctc ttacatgaaa cttctgagga tgctgaggttgctggttatt tcattccaaa 1260 gcaaacaagg gtgatgatca atgcttatgc tattgggagagacaagaatt catgggaaga 1320 tcctgatgct tttaagcctt caaggttttt gaaaccaggggtgcctgatt ttaaagggaa 1380 tcactttgag tttattcctt tcgggtctgg tcggaggtcttgccccggta tgcagcttgg 1440 gttatacaca cttgatttgg ctgttgctca cttgcttcattgttttacat gggaattgcc 1500 tgatggcatg aaaccgagtg aacttgacat gactgatatgtttggactca ccgcgccaag 1560 agcaactcga ctcgttgccg ttccgagcaa gcgtgtgctctgtcctctct aaggaaggga 1620 aaaaggtaag ggatggaaat gaatgggatt cccttctttcgtggattcta tacagaattg 1680 aggccatggt gacaaagggt caatttgcag gtttttttttttatatatat atatatataa 1740 ttgggttaaa aaaaaaaaaa aaaa 1764 4 514 PRTaspen populus tremuloides 4 Met Asp Ser Leu Val Gln Ser Leu Gln Ala SerPro Met Ser Leu Phe 1 5 10 15 Leu Ile Val Ile Ser Ser Leu Phe Phe PheGly Leu Leu Ser Arg Leu 20 25 30 Arg Arg Arg Leu Pro Tyr Pro Pro Gly ProLys Gly Leu Pro Leu Val 35 40 45 Gly Ser Met His Met Met Asp Gln Ile ThrHis Arg Gly Leu Ala Lys 50 55 60 Leu Ala Lys Gln Tyr Gly Gly Leu Phe HisMet Arg Met Gly Tyr Leu 65 70 75 80 His Met Val Thr Val Ser Ser Pro GluIle Ala Arg Gln Val Leu Gln 85 90 95 Val Gln Asp Asn Ile Phe Ser Asn ArgPro Ala Asn Ile Ala Ile Ser 100 105 110 Tyr Leu Thr Tyr Asp Arg Ala AspMet Ala Phe Ala His Tyr Gly Pro 115 120 125 Phe Trp Arg Gln Met Arg LysLeu Cys Val Met Lys Leu Phe Ser Arg 130 135 140 Lys Arg Ala Glu Ser TrpGlu Ser Val Arg Asp Glu Val Asp Ser Met 145 150 155 160 Leu Lys Thr ValGlu Ala Asn Ile Gly Lys Pro Val Asn Leu Gly Glu 165 170 175 Leu Ile PheThr Leu Thr Met Asn Ile Thr Tyr Arg Ala Ala Phe Gly 180 185 190 Ala LysAsn Glu Gly Gln Asp Glu Phe Ile Lys Ile Leu Gln Glu Phe 195 200 205 SerLys Leu Phe Gly Ala Phe Asn Met Ser Asp Phe Ile Pro Trp Leu 210 215 220Gly Trp Ile Asp Pro Gln Gly Leu Ser Ala Arg Leu Val Lys Ala Arg 225 230235 240 Lys Ala Leu Asp Arg Phe Ile Asp Ser Ile Ile Asp Asp His Ile Gln245 250 255 Lys Arg Lys Gln Asn Lys Phe Ser Glu Asp Ala Glu Thr Asp MetVal 260 265 270 Asp Asp Met Leu Ala Phe Tyr Gly Glu Glu Ala Arg Lys ValAsp Glu 275 280 285 Ser Asp Asp Leu Gln Lys Ala Ile Ser Leu Thr Lys AspAsn Ile Lys 290 295 300 Ala Ile Ile Met Asp Val Met Phe Gly Gly Thr GluThr Val Ala Ser 305 310 315 320 Ala Ile Glu Trp Val Met Ala Glu Leu MetLys Ser Pro Glu Asp Gln 325 330 335 Lys Arg Val Gln Gln Glu Leu Ala GluVal Val Gly Leu Glu Arg Arg 340 345 350 Val Glu Glu Ser Asp Ile Asp LysLeu Thr Phe Leu Lys Cys Ala Leu 355 360 365 Lys Glu Thr Leu Arg Met HisPro Pro Ile Pro Leu Leu Leu His Glu 370 375 380 Thr Ser Glu Asp Ala GluVal Ala Gly Tyr Phe Ile Pro Lys Gln Thr 385 390 395 400 Arg Val Met IleAsn Ala Tyr Ala Ile Gly Arg Asp Lys Asn Ser Trp 405 410 415 Glu Asp ProAsp Ala Phe Lys Pro Ser Arg Phe Leu Lys Pro Gly Val 420 425 430 Pro AspPhe Lys Gly Asn His Phe Glu Phe Ile Pro Phe Gly Ser Gly 435 440 445 ArgArg Ser Cys Pro Gly Met Gln Leu Gly Leu Tyr Thr Leu Asp Leu 450 455 460Ala Val Ala His Leu Leu His Cys Phe Thr Trp Glu Leu Pro Asp Gly 465 470475 480 Met Lys Pro Ser Glu Leu Asp Met Thr Asp Met Phe Gly Leu Thr Ala485 490 495 Pro Arg Ala Thr Arg Leu Val Ala Val Pro Ser Lys Arg Val LeuCys 500 505 510 Pro Leu 5 1503 DNA aspen populus tremuloidesmisc_feature AldOMT; GenBank accession number X62096 5 tcacttcctttccttacacc ttcttcaacc ttttgtttcc ttgtagaatt caatctcgat 60 caagatgggttcaacaggtg aaactcagat gactccaact caggtatcag atgaagaggc 120 acacctctttgccatgcaac tagccagtgc ttcagttcta ccaatgatcc tcaaaacagc 180 cattgaactcgaccttcttg aaatcatggc taaagctggc cctggtgctt tcttgtccac 240 atctgagatagcttctcacc tccctaccaa aaaccctgat gcgcctgtca tgttagaccg 300 tatcctgcgcctcctggcta gctactccat tcttacctgc tctctgaaag atcttcctga 360 tgggaaggttgagagactgt atggcctcgc tcctgtttgt aaattcttga ccaagaacga 420 ggacggtgtctctgtcagcc ctctctgtct catgaaccag gacaaggtcc tcatggaaag 480 ctggtattatttgaaagatg caattcttga tggaggaatt ccatttaaca aggcctatgg 540 gatgactgcatttgaatatc atggcacgga tccaagattc aacaaggtct tcaacaaggg 600 aatgtctgaccactctacca ttaccatgaa gaagattctt gagacctaca aaggctttga 660 aggcctcacgtccttggtgg atgttggtgg tgggactgga gccgtcgtta acaccatcgt 720 ctctaaatacccttcaatca agggcattaa cttcgatctg ccccacgtca ttgaggatgc 780 cccatcttatcccggagtgg agcatgttgg tggcgacatg tttgttagtg tgcccaaagc 840 agatgccgttttcatgaagt ggatatgcca tgattggagc gacgcccact gcttaaaatt 900 cttgaagaattgctatgacg cgttgccgga aaacggcaag gtgatacttg ttgagtgcat 960 tcttcccgtggctcctgaca caagccttgc caccaaggga gtcgtgcacg ttgatgtcat 1020 catgctggcgcacaaccccg gtgggaaaga gaggaccgag aaggaatttg agggcttagc 1080 taagggagctggcttccaag gttttgaagt aatgtgctgt gcattcaaca cacatgtcat 1140 tgaattccgcaagaaggcct aaggcccatg tccaagctcc aagttacttg gggttttgca 1200 gacaacgttgctgctgtctc tgcgtttgat gtttctgatt gctttttttt atacgaggag 1260 tagctatctcttatgaaaca tgtaaggata agattgcgtt ttgtatgcct gattttctca 1320 aataacttcactgcctccct caaaattctt aatacatgtg aaaagatttc ctattggcct 1380 tctgcttcaaacagtaaaga cttctgtaac ggaaaagaaa gcaattcatg atgtatgtat 1440 cttgcaagattatgagtatt gttctaagca ttaagtgatt gttcaaaaaa aaaaaaaaaa 1500 aaa 1503 6365 PRT aspen populus tremuloides 6 Met Gly Ser Thr Gly Glu Thr Gln MetThr Pro Thr Gln Val Ser Asp 1 5 10 15 Glu Glu Ala His Leu Phe Ala MetGln Leu Ala Ser Ala Ser Val Leu 20 25 30 Pro Met Ile Leu Lys Thr Ala IleGlu Leu Asp Leu Leu Glu Ile Met 35 40 45 Ala Lys Ala Gly Pro Gly Ala PheLeu Ser Thr Ser Glu Ile Ala Ser 50 55 60 His Leu Pro Thr Lys Asn Pro AspAla Pro Val Met Leu Asp Arg Ile 65 70 75 80 Leu Arg Leu Leu Ala Ser TyrSer Ile Leu Thr Cys Ser Leu Lys Asp 85 90 95 Leu Pro Asp Gly Lys Val GluArg Leu Tyr Gly Leu Ala Pro Val Cys 100 105 110 Lys Phe Leu Thr Lys AsnGlu Asp Gly Val Ser Val Ser Pro Leu Cys 115 120 125 Leu Met Asn Gln AspLys Val Leu Met Glu Ser Trp Tyr Tyr Leu Lys 130 135 140 Asp Ala Ile LeuAsp Gly Gly Ile Pro Phe Asn Lys Ala Tyr Gly Met 145 150 155 160 Thr AlaPhe Glu Tyr His Gly Thr Asp Pro Arg Phe Asn Lys Val Phe 165 170 175 AsnLys Gly Met Ser Asp His Ser Thr Ile Thr Met Lys Lys Ile Leu 180 185 190Glu Thr Tyr Lys Gly Phe Glu Gly Leu Thr Ser Leu Val Asp Val Gly 195 200205 Gly Gly Thr Gly Ala Val Val Asn Thr Ile Val Ser Lys Tyr Pro Ser 210215 220 Ile Lys Gly Ile Asn Phe Asp Leu Pro His Val Ile Glu Asp Ala Pro225 230 235 240 Ser Tyr Pro Gly Val Glu His Val Gly Gly Asp Met Phe ValSer Val 245 250 255 Pro Lys Ala Asp Ala Val Phe Met Lys Trp Ile Cys HisAsp Trp Ser 260 265 270 Asp Ala His Cys Leu Lys Phe Leu Lys Asn Cys TyrAsp Ala Leu Pro 275 280 285 Glu Asn Gly Lys Val Ile Leu Val Glu Cys IleLeu Pro Val Ala Pro 290 295 300 Asp Thr Ser Leu Ala Thr Lys Gly Val ValHis Val Asp Val Ile Met 305 310 315 320 Leu Ala His Asn Pro Gly Gly LysGlu Arg Thr Glu Lys Glu Phe Glu 325 330 335 Gly Leu Ala Lys Gly Ala GlyPhe Gln Gly Phe Glu Val Met Cys Cys 340 345 350 Ala Phe Asn Thr His ValIle Glu Phe Arg Lys Lys Ala 355 360 365 7 1915 DNA aspen populustremuloides misc_feature 4CL 7 ccctcgcgaa actccgaaaa cagagagcacctaaaactca ccatctctcc ctctgcatct 60 ttagcccgca atggacgcca caatgaatccacaagaattc atctttcgct caaaattacc 120 agacatctac atcccgaaaa accttcccctgcattcatac gttcttgaga acttgtctaa 180 acattcatca aaaccttgcc tgataaatggcgcgaatgga gatgtctaca cctatgctga 240 tgttgagctc acagcaagaa gagttgcttctggtctgaac aagattggta ttcaacaagg 300 tgacgtgatc atgctcttcc taccaagttcacctgaattc gtgcttgctt tcctaggcgc 360 ttcacacaga ggtgccatga tcactgctgccaatcctttc tccacccctg cagagctagc 420 aaaacatgcc aaggcctcga gagcaaagcttctgataaca caggcttgtt actacgagaa 480 ggttaaagat tttgcccgag aaagtgatgttaaggtcatg tgcgtggact ctgccccgga 540 cggtgcttca cttttcagag ctcacacacaggcagacgaa aatgaagtgc ctcaggtcga 600 cattagtcct gatgatgtcg tagcattgccttattcatca gggactacag ggttgccaaa 660 aggggtcatg ttaacgcaca aagggctaataaccagtgtg gctcaacagg tagatggaga 720 caatcctaac ctgtattttc acagtgaagatgtgattctg tgtgtgcttc ctatgttcca 780 tatctatgct ctgaattcaa tgatgctctgtggtctgaga gttggtgcct cgattttgat 840 aatgccaaag tttgagattg gttctttgctgggattgatt gagaagtaca aggtatctat 900 agcaccagtt gttccacctg tgatgatggcaattgctaag tcacctgatc ttgacaagca 960 tgacctgtct tctttgagga tgataaaatctggaggggct ccattgggca aggaacttga 1020 agatactgtc agagctaagt ttcctcaggctagacttggt cagggatatg gaatgaccga 1080 ggcaggacct gttctagcaa tgtgcttggcatttgccaag gaaccattcg acataaaacc 1140 aggtgcatgt ggaactgtag tcaggaatgcagagatgaag attgttgacc cagaaacagg 1200 ggtctctcta ccgaggaacc agcctggtgagatctgcatc cggggtgatc agatcatgaa 1260 aggatatctt aatgaccccg aggcaacctcaagaacaata gacaaagaag gatggctgca 1320 cacaggcgat atcggctaca ttgatgatgatgatgagctt ttcatcgttg acagattgaa 1380 ggaattgatc aagtataaag ggtttcaggttgctcctact gaactcgaag ctttgttaat 1440 agcccatcca gagatatccg atgctgctgtagtaggattg aaagatgagg atgcgggaga 1500 agttcctgtt gcatttgtag tgaaatcagaaaagtctcag gccaccgaag atgaaattaa 1560 gcagtatatt tcaaaacagg tgatcttctacaagagaata aaacgagttt tcttcattga 1620 agcaattccc aaggcaccat caggcaagatcctgaggaag aatctgaaag agaagttgcc 1680 aggcatataa ctgaagatgt tactgaacatttaaccctct gtcttatttc tttaatactt 1740 gcgaatcatt gtagtgttga accaagcatgcttggaaaag acacgtaccc aacgtaagac 1800 agttactgtt cctagtatac aagctctttaatgttcgttt tgaacttggg aaaacataag 1860 ttctcctgtc gccatatgga gtaattcaattgaatatttt ggtttcttta atgat 1915 8 1395 DNA aspen populus tremuloidesmisc_feature CAD; GenBank accession number AF217957 8 aaactccatccctctctctt agcctcgttg tttcaagaaa atgggtagcc ttgaaacaga 60 gagaaaaattgtaggatggg cagcaacaga ctcaactggg catctcgctc cttacaccta 120 tagtctcagagatacggggc cagaagatgt tcttatcaag gttatcagct gtggaatttg 180 ccataccgatatccaccaaa tcaaaaatga tcttggcatg tcacactatc ctatggtccc 240 tggccatgaagtggttggtg aggttgttga ggtgggatca gatgtgacaa agttcaaagc 300 tggagatgttgttggtgttg gagtcatcgt tggaagctgc aagaattgtc atccatgcaa 360 atcagagcttgagcaatact gcaacaagaa aatctggtct tacaatgatg tctacactga 420 tggcaaacccacccaaggag gctttgctga atccatggtt gtcgatcaaa agtttgtggt 480 gagaattcctgatgggatgt caccagaaca agcagcgccg ctgttgtgcg ctggattgac 540 agtttacagcccactcaaac actttggact gaaacagagt gggctaagag gagggatttt 600 aggacttggaggagtagggc acatgggggt gaagatagca aaggcaatgg gacaccatgt 660 aactgtgattagttcttctg acaagaagcg ggaggaggct atggaacatc ttggtgctga 720 tgaatacctggtcagctcgg atgtggaaag catgcaaaaa gctgctgatc aacttgacta 780 tatcatcgatactgtgcctg tggttcaccc tctcgagcct tacctttctc tattgaaact 840 tgatggcaagctgatcttga tgggtgttat taatacccca ttgcagtttg tttcgccaat 900 ggttatgcttgggagaaagt cgatcaccgg gagcttcata gggagcatga aggagacaga 960 ggagatgcttgagttctgca aggaaaaggg attggcctcc atgattgaag tgatcaaaat 1020 ggattatatcaacacagcat tcgagaggct tgagaaaaat gatgtgagat atagattcgt 1080 tgtcgatgttgctggtagca agcttattcc ctgaacgaca ataccattca tattcgaaaa 1140 aacgcgatatacattgatac ctgtttcaga cttgacttta ttttcgagtg atgtgttttg 1200 tggttcaaatgtgacagttt gtctttgctt ttaaaataaa gaaaaagttg agttgttttt 1260 ttattttcattaatgggcat gcgttacctt gtaattgaat gcgctgcatc tggtgatctg 1320 tcccataaactaatctcttg tggcaatgaa agatgacgaa ctttctgaaa aaaaaaaaaa 1380 aaaaaaaaaaaaaaa 1395 9 357 PRT aspen populus tremuloides 9 Met Gly Ser Leu Glu ThrGlu Arg Lys Ile Val Gly Trp Ala Ala Thr 1 5 10 15 Asp Ser Thr Gly HisLeu Ala Pro Tyr Thr Tyr Ser Leu Arg Asp Thr 20 25 30 Gly Pro Glu Asp ValLeu Ile Lys Val Ile Ser Cys Gly Ile Cys His 35 40 45 Thr Asp Ile His GlnIle Lys Asn Asp Leu Gly Met Ser His Tyr Pro 50 55 60 Met Val Pro Gly HisGlu Val Val Gly Glu Val Val Glu Val Gly Ser 65 70 75 80 Asp Val Thr LysPhe Lys Ala Gly Asp Val Val Gly Val Gly Val Ile 85 90 95 Val Gly Ser CysLys Asn Cys His Pro Cys Lys Ser Glu Leu Glu Gln 100 105 110 Tyr Cys AsnLys Lys Ile Trp Ser Tyr Asn Asp Val Tyr Thr Asp Gly 115 120 125 Lys ProThr Gln Gly Gly Phe Ala Glu Ser Met Val Val Asp Gln Lys 130 135 140 PheVal Val Arg Ile Pro Asp Gly Met Ser Pro Glu Gln Ala Ala Pro 145 150 155160 Leu Leu Cys Ala Gly Leu Thr Val Tyr Ser Pro Leu Lys His Phe Gly 165170 175 Leu Lys Gln Ser Gly Leu Arg Gly Gly Ile Leu Gly Leu Gly Gly Val180 185 190 Gly His Met Gly Val Lys Ile Ala Lys Ala Met Gly His His ValThr 195 200 205 Val Ile Ser Ser Ser Asp Lys Lys Arg Glu Glu Ala Met GluHis Leu 210 215 220 Gly Ala Asp Glu Tyr Leu Val Ser Ser Asp Val Glu SerMet Gln Lys 225 230 235 240 Ala Ala Asp Gln Leu Asp Tyr Ile Ile Asp ThrVal Pro Val Val His 245 250 255 Pro Leu Glu Pro Tyr Leu Ser Leu Leu LysLeu Asp Gly Lys Leu Ile 260 265 270 Leu Met Gly Val Ile Asn Thr Pro LeuGln Phe Val Ser Pro Met Val 275 280 285 Met Leu Gly Arg Lys Ser Ile ThrGly Ser Phe Ile Gly Ser Met Lys 290 295 300 Glu Thr Glu Glu Met Leu GluPhe Cys Lys Glu Lys Gly Leu Ala Ser 305 310 315 320 Met Ile Glu Val IleLys Met Asp Tyr Ile Asn Thr Ala Phe Glu Arg 325 330 335 Leu Glu Lys AsnAsp Val Arg Tyr Arg Phe Val Val Asp Val Ala Gly 340 345 350 Ser Lys LeuIle Pro 355 10 535 PRT aspen populus tremuloides 10 Met Asn Pro Gln GluPhe Ile Phe Arg Ser Lys Leu Pro Asp Ile Tyr 1 5 10 15 Ile Pro Lys AsnLeu Pro Leu His Ser Tyr Val Leu Glu Asn Leu Ser 20 25 30 Lys His Ser SerLys Pro Cys Leu Ile Asn Gly Ala Asn Gly Asp Val 35 40 45 Tyr Thr Tyr AlaAsp Val Glu Leu Thr Ala Arg Arg Val Ala Ser Gly 50 55 60 Leu Asn Lys IleGly Ile Gln Gln Gly Asp Val Ile Met Leu Phe Leu 65 70 75 80 Pro Ser SerPro Glu Phe Val Leu Ala Phe Leu Gly Ala Ser His Arg 85 90 95 Gly Ala MetIle Thr Ala Ala Asn Pro Phe Ser Thr Pro Ala Glu Leu 100 105 110 Ala LysHis Ala Lys Ala Ser Arg Ala Lys Leu Leu Ile Thr Gln Ala 115 120 125 CysTyr Tyr Glu Lys Val Lys Asp Phe Ala Arg Glu Ser Asp Val Lys 130 135 140Val Met Cys Val Asp Ser Ala Pro Asp Gly Ala Ser Leu Phe Arg Ala 145 150155 160 His Thr Gln Ala Asp Glu Asn Glu Val Pro Gln Val Asp Ile Ser Pro165 170 175 Asp Asp Val Val Ala Leu Pro Tyr Ser Ser Gly Thr Thr Gly LeuPro 180 185 190 Lys Gly Val Met Leu Thr His Lys Gly Leu Ile Thr Ser ValAla Gln 195 200 205 Gln Val Asp Gly Asp Asn Pro Asn Leu Tyr Phe His SerGlu Asp Val 210 215 220 Ile Leu Cys Val Leu Pro Met Phe His Ile Tyr AlaLeu Asn Ser Met 225 230 235 240 Met Leu Cys Gly Leu Arg Val Gly Ala SerIle Leu Ile Met Pro Lys 245 250 255 Phe Glu Ile Gly Ser Leu Leu Gly LeuIle Glu Lys Tyr Lys Val Ser 260 265 270 Ile Ala Pro Val Val Pro Pro ValMet Met Ala Ile Ala Lys Ser Pro 275 280 285 Asp Leu Asp Lys His Asp LeuSer Ser Leu Arg Met Ile Lys Ser Gly 290 295 300 Gly Ala Pro Leu Gly LysGlu Leu Glu Asp Thr Val Arg Ala Lys Phe 305 310 315 320 Pro Gln Ala ArgLeu Gly Gln Gly Tyr Gly Met Thr Glu Ala Gly Pro 325 330 335 Val Leu AlaMet Cys Leu Ala Phe Ala Lys Glu Pro Phe Asp Ile Lys 340 345 350 Pro GlyAla Cys Gly Thr Val Val Arg Asn Ala Glu Met Lys Ile Val 355 360 365 AspPro Glu Thr Gly Val Ser Leu Pro Arg Asn Gln Pro Gly Glu Ile 370 375 380Cys Ile Arg Gly Asp Gln Ile Met Lys Gly Tyr Leu Asn Asp Pro Glu 385 390395 400 Ala Thr Ser Arg Thr Ile Asp Lys Glu Gly Trp Leu His Thr Gly Asp405 410 415 Ile Gly Tyr Ile Asp Asp Asp Asp Glu Leu Phe Ile Val Asp ArgLeu 420 425 430 Lys Glu Leu Ile Lys Tyr Lys Gly Phe Gln Val Ala Pro ThrGlu Leu 435 440 445 Glu Ala Leu Leu Ile Ala His Pro Glu Ile Ser Asp AlaAla Val Val 450 455 460 Gly Leu Lys Asp Glu Asp Ala Gly Glu Val Pro ValAla Phe Val Val 465 470 475 480 Lys Ser Glu Lys Ser Gln Ala Thr Glu AspGlu Ile Lys Gln Tyr Ile 485 490 495 Ser Lys Gln Val Ile Phe Tyr Lys ArgIle Lys Arg Val Phe Phe Ile 500 505 510 Glu Ala Ile Pro Lys Ala Pro SerGly Lys Ile Leu Arg Lys Asn Leu 515 520 525 Lys Glu Lys Leu Pro Gly Ile530 535 11 20 DNA aspen populus tremuloides misc_feature Pt4CL1 promotersense primer 11 caggaatgct ctgcactctg 20 12 20 DNA aspen populustremuloides misc_feature Pt4CL1 sense primer 12 atgaatccac aagaattcat 2013 20 DNA aspen populus tremuloides misc_feature Pt4CL1 promoter senseprimer 13 caggaatgct ctgcactctg 20 14 21 DNA aspen populus tremuloidesmisc_feature PtCal5H antisense primer 14 ttagagagga cagagcacac g 21

What is claimed is:
 1. A method of genetically transforming a plantsimultaneously with multiple genes from the phenylpropanoid pathways,comprising incorporating into the genome of the plant a plurality ofgenes, the genes selected from the group consisting of 4CL, CAld5H,AldOMT, CAD, and SAD, substantially similar fragments thereof, andcombinations thereof to produce plants displaying altered agronomictraits.
 2. The method of claim 1 wherein the genes incorporated into thegenome are CAld5H, AldOMT, and SAD genes, substantially similarfragments thereof or combinations thereof in sense orientation, toproduce increased syringyl lignin in the plant compared to anon-transformed plant.
 3. The method of claim 1, wherein the genesincorporated into its genome are 4CL gene or substantially similarfragments thereof in a sense or antisense orientation and CAld5H,AldOMT, and SAD genes, substantially similar fragments thereof orcombinations thereof in sense orientation to downregulate 4CL geneexpression in the plant compared to a non-transformed plant.
 4. Themethod of claim 3 wherein the down regulation of 4CL correlates withdecreased lignin content, increased syringyl/guaiacyl (S/G) lignin ratioand increased cellulose content compared to a non-transformed plant. 5.The method of claim 1, wherein the genes incorporated into its genomeare 4CL and CAD genes or substantially similar fragments thereof insense or antisense orientation and CAld5H, AldOMT, and SAD genes,substantially similar fragments thereof or combinations thereof in senseorientation to downregulate 4CL and CAD gene expression in the plantcompared to a non-transformed plant.
 6. The method of claim 5 whereinthe down regulation of 4CL and CAD correlates with decreased lignincontent, increased S/G lignin ratio and increased cellulose content ascompared to a non-transformed plant.
 7. The method of claim 1 whereinthe gene incorporated into the genome of the plant is 4CL gene orsubstantially similar fragments thereof in the sense orientation, toupregulate 4CL gene expression in the plant compared to anon-transformed plant.
 8. The method of claim 7, wherein theupregulation of 4CL gene correlates to increased lignin content in theplant compared to a non-transformed plant.
 9. A method of claim 1wherein the genes incorporated into the genome of the plant are 4CL,CAld5H, AldOMT, and SAD genes, substantially similar fragments thereofor combinations thereof in the sense orientation, to upregulate 4CL,CAld5H, AldOMT, and SAD gene expression
 10. The method of claim 9wherein the upregulation of 4CL, CAld5H, AldOMT, and SAD genescorrelates to an increased lignin content and increased S/G ratiocompared to a non-transformed plant.
 11. The method of claim 1 whereinthe genes incorporated into the genome of the plant 4CL, CAld5H, AldOMT,and SAD genes, substantially similar fragments thereof or combinationsthereof in the sense orientation and the CAD gene or substantiallysimilar fragments thereof in the antisense orientation, to upregulate4CL, CAld5H, AldOMT, and SAD gene expression and to downregulate CADgene expression in the plant compared to a non-transformed plant. 12.The method of claim 11, wherein the upregulation of 4CL, CAld5H, AldOMT,and SAD gene expression and the downregulation of CAD gene expressioncorrelate with an increased lignin content and an increased S/G ratiocompared to a non-transformed plant.
 13. The method of claim 2 whereinthe plant is an angiosperm or a gymnosperm.
 14. The method of claim 3wherein the plant is an angiosperm or a gymnosperm.
 15. The method ofclaim 5 wherein the plant is an angiosperm or a gymnosperm.
 16. A methodof preparing plant cells having in their genome a plurality of DNAconstructs, the method comprising a) incorporating into the genome ofthe cells a plurality of DNA constructs to yield transformed cells, eachconstruct comprising a polynucleotide sequence encoding a proteinselected from the group consisting of 4CL, CAld5H, AldOMT, CAD, and SAD,substantially similar fragments thereof, and combinations thereof,operably linked to a promoter sequence functional in the cells, and atermination sequence b) identifying the transformed plant cells, thegenome of which is augmented with DNA from the different DNA constructs.17. The method of claim 16 wherein the expression of the protein isassociated with an agronomic trait in the plant cells.
 18. The method ofclaim 17 wherein the trait is lignin biosynthesis, cellulosebiosynthesis, growth, wood quality, stress resistance, sterility, grainyield or nutritional value.
 19. The method of claim 16 furthercomprising regenerating the identified transformed plants cells to yielda transgenic plant.
 20. The method of claim 16 wherein the plant cellsare regenerable.
 21. The method of claim 16 wherein the plant cells aretree cells.
 22. The method of claim 16 wherein the plant cells areangiosperm cells.
 23. The method of claim 16 wherein the plant cells aregymnosperm cells.
 24. A method of preparing transgenic plants havingaltered lignin and cellulose compositions, the method comprising a)providing the genome of the plants with a plurality of DNA constructs toyield transformed plant cells, each construct comprising apolynucleotide sequence encoding a protein selected from the groupconsisting of 4CL, CAld5H, AldOMT, CAD, and SAD, substantially similarfragments thereof, and combinations thereof, the polynucleotide sequenceoperably linked to a promoter sequence functional in the plant cells,and a termination sequence; b) regenerating the transformed plant cellsto yield transgenic plants, the genome of which is augmented with DNAfrom different DNA constructs, and c) expressing the DNA constructs inthe cells of the transgenic plants in an amount effective to alter thelignin and cellulose composition in the plants.
 25. A plant of claim 24wherein the promoter sequence can be constitutive or tissue-specific.26. A plant of claim 24 wherein the promoter sequence can be homologousor heterologous.
 27. A plant if claim 24 wherein the promoter sequenceprovides for transcription in xylem.
 28. A method of claim 24, whereinthe plant is a plant cell, plant organ, or an entire plant.
 29. A methodof claim 24, wherein the plant is a plant fruit, seeds and progenythereof.
 30. The method of claim 24 wherein the plants are trees. 31.The method of claim 24 wherein the plants are angiosperms.
 32. Themethod of claim 24 wherein the plants are gymnosperms.
 33. A transgenicplant prepared by the method of claim
 24. 34. A progeny plant of thetransgenic plant of claim
 32. 35. A method of preparing a transgenictree comprising a) incorporating into the genome of the tree a pluralityof desired DNA constructs to produce transformed tree cells, eachconstruct comprising a polynucleotide sequence encoding a proteinselected from the group consisting of 4CL, CAld5H, AldOMT, CAD, and SAD,substantially similar fragments thereof, and combinations thereof,operably linked to a promoter sequence functional in the cells, and atermination sequence; b) regenerating the transformed tree cells toyield transgenic trees, the genome of which is augmented with theplurality of DNA constructs; and c) expressing the DNA construct in thecells of the transgenic tree in an amount effective to alter the ligninand cellulose composition of the tree.
 36. A transgenic tree prepared bythe method of claim
 34. 37. The method of claim 34 wherein thetransgenic tree is a Populus tremuloides.
 38. A plant havingincorporated into its genome a DNA construct comprising a polynucleotidesequence encoding a protein selected from the group consisting of 4CL,CAld5H, AldOMT, CAD, and SAD, substantially similar fragments thereof,and combinations thereof, operably linked to a promoter sequence, and atermination sequence.
 39. A plant of claim 38 wherein the promotersequence can be constitutive or tissue-specific.
 40. A plant of claim 38wherein the promoter sequence can be homologous or heterologous.
 41. Aplant of claim 38 wherein the promoter sequence provides fortranscription in xylem.
 42. The plant of claim 38 which is a tree.
 43. Aplant of claim 38 wherein the plant is a gymnosperm.
 44. The plant ofclaim 43 wherein the nucleotide sequence encodes CAld5H, AldOMT, and SADgenes, substantially similar fragments thereof and combinations thereof,in sense orientation, yielding increased syringyl lignin as compared toa non-transformed plant.
 45. The plant of claim 43 wherein thenucleotide sequences encodes 4CL gene or substantially similar fragmentsthereof in sense or antisense orientation and CAld5H, AldOMT, and SADgenes, substantially similar fragments thereof, and combinationsthereof, in sense orientation, yielding decreased lignin content,increased syringyl/guaiacyl (S/G) lignin ratio and increased cellulosecontent as compared to a non-transformed plant.
 46. The plant of claim43 wherein the nucleotide sequences encodes 4CL and CAD genes orsubstantially similar fragments thereof in sense or antisenseorientation and CAld5H, AldOMT, and SAD genes, substantially similarfragments thereof, and combinations thereof, in sense orientation,yielding decreased lignin content, increased S/G lignin ratio andincreased cellulose content as compared to a non-transformed plant. 47.The plant of claim 43 wherein the nucleotide sequences encodes 4CL geneor substantially similar fragments thereof in the sense orientationyielding increased lignin content as compared to a non-transformedplant.
 48. The plant of claim 43 wherein the polynucleotide sequencesincorporated into the genome are 4CL, CAld5H, AldOMT, and SAD genes,substantially similar fragments thereof, and combinations thereof, inthe sense orientation, yielding increased lignin content and increasedS/G ratio as compared to a non-transformed plant.
 49. The plant of claim43 wherein the polynucleotide sequences encodes 4CL, CAld5H, AldOMT, andSAD genes, substantially similar fragments thereof and combinationsthereof in the sense orientation and the CAD gene, or substantiallysimilar fragments thereof in the antisense orientation, yieldingincreased lignin content and increased S/G ratio as compared to anon-transformed plant.
 50. A plant of claim 38 wherein the plant is anangiosperm.
 51. The plant of claim 50 wherein the polynucleotidesequences encodes CAld5H, AldOMT, and SAD genes, substantially similarfragments thereof and combinations thereof, in sense orientation,yielding increased S/G lignin ratio as compared to a non-transformedplant.
 52. The plant of claim 51 wherein the polynucleotide sequencesencode the 4CL gene or substantially similar fragments thereof in senseor antisense orientation, and CAld5H, AldOMT, and SAD genes,substantially similar fragment thereof, and combinations thereof insense orientation, yielding decreased lignin content, increased S/Glignin ratio and increased cellulose content as compared to anon-transformed plant.
 53. The plant of claim 50 wherein the nucleotidesequences encodes 4CL and CAD genes or substantially similar fragmentsthereof, and combinations thereof by sense or antisense orientation andCAld5H, AldOMT, and SAD genes, substantially similar fragments thereof,and combinations thereof in sense orientation, yielding decreased lignincontent, increased S/G lignin ratio and increased cellulose content ascompared to a non-transformed plant.
 54. The plant of claim 50 whereinthe nucleotide sequences encodes 4CL gene or substantially similarfragment thereof, by sense orientation, yielding increased lignincontent as compared to a non-transformed plant.
 55. The plant of claim50 wherein the nucleotide sequences encodes 4CL, CAld5H, AldOMT, and SADgenes, substantially similar fragment thereof, and combinations thereof,in sense orientation, yielding increased lignin content and increasedS/G ratio as compared to a non-transformed plant.
 56. The plant of claim50 wherein the nucleotide sequences encode 4CL CAld5H, AldOMT, and SADgenes, substantially similar fragment thereof, and combinations thereof,in sense orientation, and CAD gene, substantially similar fragmentthereof, and combinations thereof in antisense orientation, yieldingincreased lignin content and increased S/G ratio as compared to anon-transformed plant.
 57. A plurality of DNA constructs, each constructcomprising in the 5′-3′ direction: a) a gene promoter sequence, b) agene termination sequence; and c) a polynucleotide sequence encoding aprotein selected from the group consisting of 4CL, CAld5H, AldOMT, CAD,and SAD, substantially similar fragments thereof, and combinationsthereof involved in a lignin biosynthetic pathway, the polynucleotidesequence operably linked to the promoter and termination sequences. 58.A plurality of DNA constructs of claim 57 wherein the gene promotersequence is a constitutive or tissue-specific promoter.
 59. A pluralityof DNA constructs of claim 57 wherein the gene promoter sequence ishomologous or heterologous.
 60. A plurality of DNA constructs of claim57 wherein the gene promoter sequences are xylem-specific.
 61. Aplurality of DNA constructs incorporated into the genome of gymnosperms,the constructs comprising nucleotide sequences encoding CAld5H, AldOMT,and SAD genes, a substantially similar fragments thereof andcombinations thereof, in sense orientation, to produce syringyl ligninas compared to a non-transformed plant.
 62. A plurality of DNAconstructs incorporated into the genome of gymnosperm, the constructscomprising 4CL gene or substantially similar fragments thereof in senseor antisense orientation and CAld5H, AldOMT, and SAD genes,substantially similar fragments thereof and combination thereof in senseorientation to decrease lignin content, increase syringyl/guaiacyl (S/G)lignin ratio and increase cellulose content as compared to anon-transformed plant.
 63. A plurality of DNA constructs incorporatedinto the genome of gymnosperms, the constructs comprising 4CL and CADgenes or substantially similar fragments thereof in sense or antisenseorientation and CAld5H, AldOMT, and SAD genes, substantially similarfragments thereof and combination thereof in sense orientation todecrease lignin content, increase S/G lignin ratio and increasecellulose content as compared to a non-transformed plant.
 64. A DNAconstruct incorporated into the genome of plants, comprising 4CL gene orsubstantially similar fragments thereof in the sense orientation toincrease lignin content compared to a non-transformed plant.
 65. A DNAconstruct of claim 63 wherein the plant is an angiosperm or agymnosperm.
 66. A plurality of DNA constructs incorporated into thegenome of plants, the constructs comprising 4CL, CAld5H, AldOMT, and SADgenes, substantially similar fragments thereof or combination thereof,in the sense orientation, to increase lignin content and increase S/Gratio compared to a non-transformed plant.
 67. A plurality of DNAconstructs of claim 65 wherein the plant is an angiosperm or agymnosperm.
 68. A plurality of DNA constructs incorporated into thegenome of plants, the constructs comprising 4CL, CAld5H, AldOMT, and SADgenes, substantially similar fragments thereof and combination thereofin the sense orientation and CAD gene or substantially similar fragmentsthereof in the antisense orientation to increase lignin content andincrease S/G ratio compared to a non-transformed plant.
 69. A pluralityof DNA constructs of claim 67 wherein the plant is an angiosperm or agymnosperm.
 70. A plurality of DNA constructs incorporated into thegenome of angiosperms, the constructs comprising CAld5H, AldOMT, and SADgenes, substantially similar fragments thereof and combinations thereofin sense orientation to engineer high S/G lignin ratio compared to anon-transformed plant.
 71. A plurality of DNA constructs incorporatedincorporating into the genome of angiosperms, the constructs comprising4CL gene or substantially similar fragment thereof in sense or antisenseorientation and CAld5H, AldOMT, and SAD genes, substantially similarfragment thereof, and combinations thereof, in sense orientation, todecrease lignin content, increase S/G lignin ratio and increasecellulose content compared to a non-transformed plant.
 72. A pluralityof DNA constructs incorporated into the genome of angiosperms, theconstructs comprising 4CL and CAD genes, substantially similar fragmentsthereof, and combinations thereof by sense or antisense orientation andCAld5H, AldOMT, and SAD genes, or substantially similar fragmentsthereof, and combinations thereof, in sense orientation, to decreaselignin content, increase S/G lignin ratio and increase cellulose contentcompared to a non-transformed plant.